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WO1996006624A1 - Proteine mutante, procedes et materiaux employes pour la produire et l'utiliser - Google Patents

Proteine mutante, procedes et materiaux employes pour la produire et l'utiliser Download PDF

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Publication number
WO1996006624A1
WO1996006624A1 PCT/US1994/009729 US9409729W WO9606624A1 WO 1996006624 A1 WO1996006624 A1 WO 1996006624A1 US 9409729 W US9409729 W US 9409729W WO 9606624 A1 WO9606624 A1 WO 9606624A1
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WIPO (PCT)
Prior art keywords
unmutated
amino acid
mutant protein
crp
subunit
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PCT/US1994/009729
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English (en)
Inventor
Lawrence A. Potempa
Hans H. Liao
Becky L. Crump
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Immtech International, Inc.
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Publication date
Application filed by Immtech International, Inc. filed Critical Immtech International, Inc.
Priority to AU12875/95A priority Critical patent/AU1287595A/en
Priority to PCT/US1994/009729 priority patent/WO1996006624A1/fr
Publication of WO1996006624A1 publication Critical patent/WO1996006624A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4737—C-reactive protein
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • This invention relates to a mutant protein having at least one of the biological activities of modified-C-reactive protein and to methods and materials for making the mutant protein by recombinant DNA techniques.
  • the invention also relates to methods and materials for using the mutant protein.
  • C-reactive protein was first described by Tillett and Francis rj. EXP. Med.. 52. 561-71 (1930)] who observed that sera from acutely ill patients precipitated with the C-polysaccharide of the cell wall of Streptococcus pneumoniae. Others subsequently identified the reactive serum factor as protein, hence the designation "C-reactive protein.”
  • CRP C-reactive protein
  • CRP binds phosphorylcholine, suggesting a role for CRP as an opsonin for microorganisms and damaged tissue that have exposed phosphorylcholine groups; (2) CRP binds chromatin, suggesting that CRP may act to scavenge chromatin released by cell lysis; (3) CRP neutralizes platelet activating factor, suggesting that CRP may function as a regulator of platelet and neutrophil activities; and (4) CRP complexed to certain other molecules or liposomes activates complement, suggesting that CRP may trigger the complement cascade. See Kaplan et al., J. Immunol.. 112. 2135-2147 (1974); Volanakis et al., J. Immunol.. 113.
  • CRP is a pentamer which consists of five identical subunits, each having a molecular weight of about 23,500.
  • the pentameric form of CRP is sometimes referred to as "native CRP.”
  • Modified-CRP has significantly different charge, size, solubility and antigenicity characteristics as compared to native CRP. Pote pa et al., Mol. Immunol.. 20. 1165-75 (1983). Modified-CRP also differs from native CRP in binding characteristics; for instance, mCRP does not bind phosphorylcholine. Id.; Chudwin et al.. J. Aller ⁇ v Clin. Immunol.. 77. 216a (1986) . Finally, mCRP differs from native CRP in its biological activity. See Potempa et al., Protides Biol. Fluids. 34. 287-290 (1986); Potempa et al., Inflammation. 12. 391-405 (1988).
  • Neo-CRP antigenicity is expressed on:
  • preCRP the primary translation product of DNA coding for CRP
  • the neo-CRP antigenicity may be detected with antibodies.
  • an antiserum made specific for neo-CRP can be used. See Potempa et al., Mol. Immunol.. 24. 531-41 (1987) .
  • the unique antigenic determinants of mCRP can be detected with monoclonal antibodies. Suitable monoclonal antibodies are described in U.S. Patent No. 5,272,257, published PCT application WO 91/00872 (published January 24, 1991; corresponding to U.S. Patent No. 5,272,257), Ying et al., J. Immunol. , 143. 221-228 (1989), Ying et al., Immunol ⁇ . 76. 324-330 (1992), and Ying et al., Molec. Immunol.. 29. 677-687 (1992) .
  • a molecule reactive with antiseru specific for neo-CRP has been identified on the surface of 10-25% of peripheral blood lymphocytes (predominantly NK and B cells) , 80% of monocytes and 60% of neutrophils, and at sites of tissue injury.
  • peripheral blood lymphocytes predominantly NK and B cells
  • mCRP can influence the development of monocyte cytotoxicity, improve the accessory cell function of monocytes, potentiate aggregated-IgG-induced phagocytic cell oxidative metabolism, and increase the production of interleukin-1, prostaglandin E and lipoxygenase products by monocytes.
  • Potempa et al. Protides Biol. Fluids. 34. 287-290 (1987); Chu et al., Proc. Aroer. Acad. Cancer Res. , 28. 344a (1987); Potempa et al., Proc. Amer. Acad. Cancer Res.. 28. 344a (1987); Zeller et al., Fed. Proc.. 46.
  • Modified-CRP has also been found to be effective in treating viral infections (see co-pending U.S. application Serial No. 08/117,874, filed September 7, 1993, a continuation of application Serial No. 07/799,448, filed November 27, 1991, now abandoned), non- Streptococcal bacterial infections and endotoxic shock (see allowed U.S. application Serial No. 07/800,508, filed November 27, 1991), and cancer (see issued U.S. Patent No. 5,283,238 and co-pending U.S. application Serial No. 08/149,663, filed November 9, 1993).
  • mCRP was preferably made using purified CRP as a starting material.
  • CRP was prepared from CRP by denaturing the CRP.
  • CRP could be denatured by: (1) treatment with an effective amount of urea (preferably 8M) in the presence of a conventional chelator (preferably ethylenediamine tetraacetic acid (EDTA) or citric acid) ; (2) adjusting the pH of the CRP to below about 3 or above about 11-12; or (3) heating CRP above 50°C for a time sufficient to cause denaturation (preferably at 63°C for 2 minutes) in the absence of calcium or in the presence of a chelator such as those listed above.
  • a conventional chelator preferably ethylenediamine tetraacetic acid (EDTA) or citric acid
  • EDTA ethylenediamine tetraacetic acid
  • Urea treatment has been the preferred method.
  • mCRP can be prepared from CRP by adsorbing the CRP onto solid surfaces. It is believed that mCRP prepared from CRP is formed by the dissociation of the five CRP subunits, each of which then undergoes a spontaneous conformational change to form mCRP. See Bray et al., Clin. Immuno1. News1etter. 8_, 137-140 (1987).
  • preCRP the primary translation product of the CRP mRNA
  • preCRP is a precursor protein consisting of a signal or leader sequence attached to the N-terminus of the CRP subunit.
  • the signal or leader sequence is cleaved from the preCRP molecule to produce mature CRP subunits which assemble into pentameric native CRP.
  • mCRP could be prepared by selecting conditions so that pentameric native CRP is not formed from the preCRP. This can be accomplished by expressing a CRP genomic or cDNA clone in a prokaryotic host. See Samols and Hu, Prot. Biol. Fluids. 34., 263-66 (1986).
  • mutant CRP subunit or preCRP molecule having the biological activities of mCRP, but which was less likely to form covalently cross-linked aggregates than the unmutated protein, would be highly desirable in order to make processing and purification easier and more efficient.
  • the present invention provides mutant proteins having these characteristics, and these mutant proteins may be produced by site-directed mutagenesis of a CRP cDNA or genomic clone as further described below.
  • Trp67 is critical for the structure of the phosphorylcholine binding site of CRP, that Lys57 and Arg58 also participate in the formation of this binding site, and that the tetrapeptide 39-Phe-Tyr- Thr-Glu has only a minimal or no role in the formation of this binding site.
  • the invention provides a mutant protein which has the same amino acid sequence as an unmutated CRP subunit or an unmutated preCRP, except that at least one amino acid of the unmutated CRP subunit or unmutated preCRP has been deleted, at least one amino acid of the unmutated CRP subunit or unmutated preCRP has been replaced by another amino acid, at least one amino acid has been added to the unmutated CRP subunit or unmutated preCRP, or a combination of such changes has been made.
  • the amino acid(s) added, deleted and/or replaced are chosen so that the mutant protein is less likely to form covalently cross-linked aggregates than the unmutated CRP subunit or unmutated preCRP.
  • the mutant protein also exhibits at least one of the biological activities of mCRP.
  • the invention further provides a DNA molecule coding for the mutant protein of the invention and a vector for expression of the mutant protein.
  • the vector comprises a DNA sequence coding for a mutant protein of the invention operatively linked to expression control sequences.
  • a host cell which has been transformed so that it contains DNA coding for a mutant protein of the invention.
  • the DNA coding for the mutant protein is operatively linked to expression control sequences.
  • the invention further provides a method of producing the mutant protein of the invention.
  • the method comprises culturing a host cell which has been transformed so that it contains DNA coding for a mutant protein.
  • the DNA coding for the mutant protein is operatively linked to expression control sequences. The culturing takes place under conditions permitting expression of the mutant protein.
  • the invention also provides methods of using the mutant proteins of the invention.
  • the mutant proteins of the invention will have at least one of the biological activities of mCRP and can be used as mCRP would be used.
  • the mutant proteins of the invention bind aggregated immunoglobulin and immune complexes. They can, therefore, like mCRP, be used to remove aggregated immunoglobulin and immune complexes from fluids, to quantitate immune complexes, and to reduce the levels of immune complexes in a mammal in need thereof.
  • the mutant proteins of the invention can also be used to treat viral infections, bacterial infections, endotoxic shock and cancer.
  • the invention further provides a device for removing aggregated immunoglobulin and immune complexes from fluids.
  • the device comprises a solid surface to which is bound a mutant protein of the invention.
  • the device also comprises a means for encasing the solid surface so that the fluid may be contacted with the solid surface.
  • the invention also provides a kit for quantitating immune complexes.
  • the kit comprises a container of one of the mutant proteins of the invention.
  • the invention provides a therapeutic composition comprising a mutant protein of the invention in combination with a pharmaceutically-acceptable carrier and a mutant protein of the invention which is labeled.
  • the labeled mutant protein may be used to detect or quantitate immune complexes or to detect cancer cells.
  • Figure IA is a diagram of a series of polymerase chain reactions.
  • Figure IB is a restriction map of plasmid pIT4.
  • Figures 2A and 2B illustrate the preparation of plasmid pIT3.
  • Figures 3A-D are elution profiles of materials chromatographed on a Q-Sepharose Fast Flow 11 (Pharmacia) column.
  • Figure 3A is native CRP
  • Figure 3B is mCRP
  • Figure 3C is wild-type recombinant CRP
  • Figure 3C is a mutant protein according to the invention.
  • Figure 4 is a PhastGel R SDS-PAGE gel (Pharmacia) which has been stained with Coomassie blue.
  • Figures 5A-B are graphs of the results of ELISA assays to detect the presence of native CRP and mCRP antigenic determinants on wild-type recombinant CRP.
  • Figures 5C-D are graphs of the results of ELISA assays to detect the presence of mCRP antigenic determinants on wild-type recombinant CRP and a mutant protein according to the invention, both of which have been purified by passage over a Q-Sepharose Fast Flow 1 column.
  • Figures 6A-B are graphs of the results of ELISA assays to detect binding to aggregated IgG and monomeric IgG.
  • Figure 7 is a diagram of a series of polymerase chain reactions.
  • Figure 8 illustrates the preparation of plasmid pIT13.
  • Figure 9 illustrates the strategy for DNA sequencing of the mutant CRP coding region of pITl3.
  • Figure 10A is an SDS-PAGE gel obtained by electrophoresing three lots of inclusion body preparations isolated from E . coli BLR(DE3) bearing plasmid pIT13.
  • Figure 10B is a Western blot obtained by using monospecific goat anti-neo-CRP antiserum LP3-HRP to stain the SDS-PAGE electrophoretic patterns of three lots of inclusion body preparations isolated from E . coli BLR(DE3) bearing plasmid pIT13.
  • Figure 11 is a representative elution profile obtained from Q-Sepharose Fast Flow 11 chromatography performed as a step in the purification of a mutant protein according to the invention from E. coli BLR(DE3) bearing plasmid pIT13.
  • Figure 12 is a representative elution profile obtained by Superdex 200 chromatography performed as a step in the purification of a mutant protein according to the invention from E. coli BLR(DE3) bearing plasmid pIT13.
  • Figure 13 is a representative elution profile obtained by Sephadex G-25 chromatography performed as a step in the purification of a mutant protein according to the invention from E. coli BLR(DE3) bearing plasmid pIT13.
  • Figure 14 presents representative SDS-PAGE results for the complete purification scheme for isolating a mutant protein according to the invention from E. coli BLR(DE3) bearing plasmid pIT13.
  • Figure 15A is an SDS-PAGE gel obtained by electrophoresing mCRP and a mutant protein according to the invention purified from E. coli BLR(DE3) bearing plasmid pIT13.
  • Figure 15B is the Western blot obtained by using anti-neo-CRP monoclonal antibody 3H12 to stain the SDS-PAGE electrophoretic patterns of mCRP and a mutant protein according to the invention purified from E. coli BLR(DE3) bearing plasmid pIT13.
  • Figures 16A-C are graphs of the results of ELISA assays performed to detect the presence of neo-CRP antigenic determinants on mCRP and a mutant protein according to the invention purified from E. coli BLR(DE3) bearing plasmid pIT13.
  • Three different anti-neo-CRP monoclonal antibodies were used: 3H12 ( Figure 17A) ; 8C10 ( Figure 17B) ; and 7A8 ( Figure 17C) .
  • Figure 17A is a graph of the results of an ELISA assay performed to detect binding of mCRP and a mutant protein according to the invention purified from E. coli BLR(DE3) bearing plasmid pIT13 to aggregated IgG.
  • Figure 17B is a graph of the results of an ELISA assay performed to detect binding of mCRP and a mutant protein according to the invention purified from E. coli BLR(DE3) bearing plasmid pIT13 to immune complexes.
  • Figure 18A is an SDS-PAGE gel obtained by electrophoresing the digests produced by treating mCRP and the mutant proteins produced by plasmids pIT4 and pIT13 with BNPS-Skatole.
  • Figure 18B is an SDS-PAGE gel obtained by electrophoresing the digests produced by treating the mutant proteins produced by plasmids pIT4 and pIT13 with endoproteinase Lys C.
  • the mutant proteins of the invention have at least one amino acid added, deleted or replaced as compared to an unmutated CRP subunit or unmutated preCRP.
  • the mutant proteins may have several amino acid changes as compared to the unmutated CRP subunit or unmutated preCRP.
  • the mutant proteins may have several added amino acids, several deleted amino acids, several replacement amino acids, or a combination of added, deleted or replacement amino acids, as compared to the unmutated CRP subunit or preCRP.
  • the amino acid(s) added, deleted and/or replaced are chosen so that the mutant protein is less likely to form covalently cross-linked aggregates than the unmutated CRP subunit or unmutated preCRP.
  • Suitable amino acid changes include the deletion or replacement of at least one, preferably all, of the cysteines in an unmutated CRP subunit or unmutated preCRP. All CRP subunits and preCRP's contain at least one cysteine. Mammalian CRP subunits contain two cysteines, and mammalian preCRP's contain three cysteines. It is believed that some of these cysteines form intermolecular disulfide bonds, thereby contributing to the formation of covalently cross-linked aggregates.
  • cysteines are desirably deleted or replaced.
  • cysteines are replaced with other amino acids, they are preferably replaced with glycine, alanine, valine, leucine, isoleucine, serine, threonine or ethionine, but any amino acid can be used. Most preferred is substitution with alanine.
  • mutant proteins of the invention are easier to purify with much higher yields than unmutated CRP subunits or unmutated preCRP 7 s. Also, the final product is much purer with many fewer aggregates and fragments than that obtained with unmutated CRP subunits or unmutated preCRP's.
  • the recombinant DNA manipulations used to produce the mutant proteins may result in amino acids being added at the amino or carboxy terminal ends of the CRP subunit or preCRP. This is acceptable as long as these amino acids do not contribute to the production of covalently cross-linked aggregates.
  • some of the amino acid changes may be made for other purposes.
  • solubility of the mutant proteins of the invention is improved if lysine residues are chemically altered by treatment with sulfo-N- hydroxysuccinimide-acetate (sulfo-NHS-acetate; which changes the positive charge of the epsilon amine group of lysine to a neutral charge), sulfosuccinimidyl-3-(4- hydroxyphenyl) propionate (which changes the positive charge of the epsilon amine group of lysine to a neutral charge and adds an aromatic functional group) , or succinic anhydride (which changes the positive charge of the epsilon amine group of lysine to a negative charge) .
  • sulfo-N- hydroxysuccinimide-acetate which changes the positive charge of the epsilon amine group of lysine to a neutral charge
  • one or more of the lysine residues of an unmutated CRP subunit or preCRP is preferably deleted or replaced with another amino acid to improve the solubility of the resultant mutant protein.
  • Other suitable amino acid changes to increase the solubility of the mutant proteins of the invention include deleting one or more hydrophobic amino acids, replacing one or more hydrophobic amino acids with charged amino acids, adding one or more charged amino acids, or combinations of these changes.
  • Aqueous media include water, saline, buffers, culture media, and body fluids.
  • the mutant proteins of the invention also exhibit at least one of the biological activities of mCRP.
  • biological activity refers to properties of mCRP other than its physical and chemical properties.
  • the biological activities of mCRP include its ability to bind aggregated immunoglobulin and immune complexes which allows mCRP to be used to removed aggregated immunoglobulin and immune complexes from fluids (such as antibody reagents or body fluids) , to quantitate immune complexes, and to reduce the levels of immune complexes in a mammal in need thereof.
  • the biological activities of mCRP also include its effectiveness in treating viral infections, bacterial infections, endotoxic shock and cancer.
  • the mutant proteins of the present invention can bind aggregated immunoglobulin and immune complexes.
  • the binding of the aggregated immunoglobulin or immune complexes may be accomplished by adding a mutant protein directly to a fluid containing aggregated immunoglobulin or immune complexes, or a mutant protein may first be immobilized on a solid support before being contacted with the fluid containing the aggregated immunoglobulin or immune complexes.
  • fluids may be incubated statically on the immobilized mutant protein when used for diagnostic assays, or fluids may be passed dynamically across the immobilized mutant protein in an extracorporeal device when used for therapeutic treatment to bind immune complexes in a body fluid.
  • Suitable solid support materials for use in the present invention may be made of agarose-based resin, polyacrylamide, polymethyl-methacrylate, polycarbonate, polysulfone, polyacrylonitrile, polyethylene, polypropylene, latex, dextran, glass, nylon, polyvinyl alcohol, gels, clay, cellulose derivatives, and any other hydrophobic or hydrophilic polymeric material.
  • the solid support may be in the form of beads for use in a column, may be in the form of the wells of a microtiter plate, may be in the form of a hollow fiber membrane, or may take other forms as discussed below.
  • Column and solid phase materials are commercially available in the United States from Bio Rad Laboratories (Richmond, CA) ; Pierce Chemical Co. (Rockford, IL) ; Pall Biosupport (Glen Cove, NY) ; Micro Membranes (Newark, NJ) ; Pharmacia Fine Chemicals (Uppsala, Sweden) ; and others.
  • mutant protein may be immobilized by covalent or non-covalent binding to the solid support.
  • Methods of immobilizing proteins on solid supports are well known in the art. For instance, mutant proteins may be immobilized on solid supports by simply incubating the mutant protein with the solid support to adsorb the mutant protein.
  • a linking agent may be utilized to secure the attachment of the mutant protein to polymeric materials.
  • the mutant protein may be immobilized non-covalently or covalently on the surface of the polymeric material with the linking agent.
  • linking agents are incorporated as part of, or derivatized onto, the polymeric solid surface before the mutant protein is added.
  • the linking agents are conventional; they include diimidoesters, carbodiimide, periodate, alkylhalides, dimethylpimelimidate and dimaleimides [See Blait, A.H., and Ghose, T.I., J. Immunol. Methods. 5_9_:129 (1983); Blair, A.H. , and Ghose, T.I., Cancer Res.. 41:2700 (1981); Gauthier, et al., J. Expr. Med.. 156:766-777 (1982) ].
  • Conjugation of the mutant protein to the linking agent generally requires an initial modification of protein amino acid R groups or cross linking agent or both. Such modifications may serve to selectively activate R groups (e.g.. carbodiimide-O-acyl urea, intermediate formation with aspartic, glutamic, and C- terminal carboxyl residues) to allow for reaction with appropriate available agent functional groups (amino groups in the case of carbodiimide) . Modifications can also include the introduction of a new reactive moiety (e.g. , N-succinimidyl-3-(2-pyridyldithio)-propionate) which introduces pyridyldithio groups on lysine epsilon- a ino residues. This allows for disulfide bond formation between protein and linking agent. In some cases, bifunctional coupling reagents are employed which form bridges between the protein R groups and the linking agent of interest.
  • R groups e.g.. carbodiimide-O-
  • the mutant proteins may be used to remove aggregated immunoglobulin or immune complexes from fluids used for research, in therapeutic procedures, or in diagnostic tests, e.g.. solutions containing monoclonal antibodies, antisera, derivatized reagents, intravenous gamma globulin, or isolated blood components.
  • the presence of aggregated immunoglobulin in such fluids may be expected because of processing steps used to make these fluids, such as heat treatment of antisera to inactivate complement.
  • the mutant protein may be bound on a solid support for removing aggregated or complexed immunoglobulins from such fluids. Suitable solid supports are those described above, and the mutant protein is bound to them in the ways described above. Alternatively, the mutant protein may be added directly to such fluids in order to remove the aggregated or complexed immunoglobulins.
  • Mutant proteins of the present invention can also be used to remove immune complexes from body fluids, such as whole blood or plasma, by contacting the fluid with the mutant protein.
  • the mutant protein may be added directly to the body fluid or may be immobilized on a solid support and then contacted with the body fluid.
  • Suitable solid supports are those described above, and the mutant protein is bound to them in the ways described above.
  • Persistent high levels of immune complexes may be found in diseases such as cancer, autoimmune diseases, arthritis, and infections.
  • the continued presence of immune complexes in the circulation and their deposition in tissues contributes to compromised immune system function and inflammatory pathology. Accordingly, reducing the level of immune complexes should be beneficial. See Theofilopoulos et al., Adv. Immunol.. 28. 90-220 (1979); Theofilopoulos et al., Immunodiagnostics of Cancer, p. 896 (1979) .
  • the mutant protein may be contacted with the body fluid by passing the blood, plasma or other fluid through an extracorporeal device having a solid support coated with mutant protein.
  • the device also will have a means for encasing the solid support so that the fluid may contact the mutant protein bound to the solid support.
  • the blood, plasma or other body fluid is circulated dynamically through the device so that the immune complexes contained therein are bound and removed as in, e.g.. conventional plasmapheresis or hemodialysis techniques.
  • the fluids can be returned to the body negating the need for blood replacement therapy.
  • the solid support and encasing means of the extracorporeal device may be made of any biocompatible material.
  • the solid support may be a membranous surface, agarose-based beads or hollow fibers coated with mutant protein.
  • the extracorporeal device may be a column packed with beads or a cylinder encasing a hollow fiber membrane.
  • the device may also include appropriate tubing for connecting it to a patient and a pump to aid the passage of the fluid through the device and back into the patient and to prevent air from entering the system.
  • conventional plasmapheresis devices may be used in the practice of the present invention by modifying them so that they contain a solid support on which mutant protein is immobilized. See, e.g.. Randerson et al., Art. Organs.
  • the device must be sterilized for therapeutic use. Sterilization may be accomplished in conventional ways such as steam, heat, purging with ethylene oxide or irradiation.
  • the invention also comprises a method of detecting or quantitating immune complexes comprising contacting the immune complexes with a mutant protein of the invention so that the immune complexes bind to the mutant protein.
  • the mutant protein may be added directly to fluids containing the immune complexes to detect or quantitate immune complexes in the fluids. Alternatively, the mutant protein may be immobilized on a solid support before contacting it with fluids containing immune complexes.
  • mutant protein may also be added directly to cells or a tissue sample having immune complexes thereon, and labeled mutant protein may be injected into a mammal so that it localizes in areas of the mammal's body where immune complexes are found, such as areas of inflammation.
  • Suitable solid support materials are those described above, and mutant protein is immobilized on them in the ways described above.
  • suitable solid supports include those conventionally used for immunoassays.
  • the solid support may be test tubes, the wells of a microtiter plate, latex beads, glass beads, other beads, filter paper, glass fiber filter paper, or dipsticks made of, e.g.. polycarbonate, polysulfone or latex.
  • labeled mutant protein can be used.
  • the labels useful in the invention are those known in the art such as enzyme, fluorescent, bioluminescent, chemiluminescent, and radioactive labels and biotin.
  • the immune complexes can be detected or quantified using conventional immunoassay techniques by adding a labeled component that binds to the immune complexes or the mutant protein.
  • Suitable labeled components include conventional reagents used in immunoassays. For instance, labeled antibodies to the immunoglobulin or the antigen in the immune complexes could be used or labeled Protein A which binds to the Fc portion of immunoglobulins could be used. The labels used are those described above.
  • Suitable conventional immunoassay techniques include agglutination, radioimmunoassay, enzyme immunoassays and fluorescence assays.
  • the mutant protein may be coated onto latex beads for use in agglutination assays or onto dipsticks for use in qualitative or semi-quantitative immunoassays.
  • Enzyme- linked immunosorbent assays are preferred since they provide a means for sensitive quantitation of levels of immune complexes.
  • any immunoassay technique can be used which results in an observable change in properties.
  • the specific concentrations of reagents, the temperatures and times of incubations, as well as other assay conditions can be varied in whatever assay is employed to detect or quantitate immune complexes depending on such factors as the concentration of the immune complexes in the sample, the nature of the sample, and the like. Those skilled in the art will be able to determine operative and optimal assay conditions for each determination while employing routine experimentation. Since body fluids from mammals normally contain immune complexes, comparison of the levels of immune complexes in a test sample from a mammal will have to be made to the levels found in normals to identify levels of immune complexes indicative of a disease state.
  • a test kit for detecting or quantitating immune complexes is also part of the invention.
  • the kit comprises a container holding a solution of mutant protein or mutant protein attached to a solid support.
  • the solid supports are the types described above, and the mutant protein is attached as described above.
  • the container could be a bottle holding a solution of mutant protein, a dipstick coated with mutant protein encased in a protective package, a bottle holding latex beads coated with mutant protein, or a microtiter plate, the wells of which are coated with mutant protein.
  • the mutant protein may be labeled if it is to be used for detecting or quantitating the immune complexes.
  • the kit may further comprise a container holding a labeled component that allows for the detection or quantitation of the immune complexes by binding to the immune complexes or to the mutant protein. Suitable labeled components were described above.
  • the mutant proteins of the invention can be administered to a mammal to reduce the level of immune complexes in the mammal. It is believed that, upon introduction of the mutant protein into a body fluid of the mammal containing the immune complexes, the soluble immune complexes will grow larger in physical size and precipitate (fall out of solution) or will otherwise be modified to enhance their removal by phagocytes. To reduce the level of immune complexes in a mammal, an effective amount of a mutant protein is administered to the mammal by injection, preferably intravenous injection.
  • Modified-CRP has been chemically altered with the following reagents and the ability of the resultant chemically-altered mCRP's to bind aggregated IgG ascertained using an ELISA assay like that described in Example 1: sulfo-NHS-acetate (alters lysines) ; sulfosuccinimidyl-3-(4-hydroxyphenyl) propionate (alters lysines) ; succinic anhydride (alters lysines) ; NHS-biotin (alters lysines) ; p-hydroxy phenylgloxal (alters arginines) ; [2-(2-nitrophenylsulfeneyl)-3-methyl-3'- bromoindoleine] (alters tryptophans) ; tetranitromethane (alters tyrosines) ; iodobeads (alters tyrosines) ; diazo- norleucine
  • tryptophan residues and amino acids with carboxylic acid groups 'e.g.. aspartic acid and glutamic acid were involved in the binding of aggregated IgG by mCRP. Accordingly, in preparing mutant proteins according to the invention for use in binding aggregated immunoglobulin or immune complexes, the tryptophan residues and the amino acids having a carboxylic acid group in the unmutated CRP subunit or preCRP should preferably not be deleted or replaced with other amino acids as any such deletion or replacement may decrease the ability of the resultant mutant protein to bind aggregated immunoglobulin or immune complexes.
  • the mutant proteins of the invention can also be used to treat viral infections. They can be used to treat any type of viral infection such as Retroviridae infections.
  • the Retroviridae are a family of spherical enveloped RNA viruses comprising three sub-families: Oncovirinae. Spurmavirinae and Lentivirinae. Hull et al, Virology: Directory & Dictionary of Animal. Bacterial and Plant viruses, page 191 (Stockton Press 1989) . Replication starts with reverse transcription of virus RNA into DNA which becomes integrated into the chromosomal DNA of the host. Id.. Endogenous oncoviruses occur widely among vertebrates and are associated with many diseases. Id. The lentiviruses include HIV-1 and SIV. Fauci, Science. 239. 617-622 (1988).
  • an effective amount of a mutant protein is administered to the mammal.
  • the mutant protein is preferably administered to the mammal before the infection becomes too serious.
  • the mutant protein is administered at the first indication of a viral infection or prophylactically to those at risk of developing viral infections.
  • the mutant protein may be administered prophylactically to hemophiliacs or surgical patients who may receive blood contaminated with a virus such as HIV-l or hepatitis.
  • a mutant protein can be administered to a mammal already suffering from a viral infection.
  • the mutant protein will generally be administered to the mammal suffering from a viral infection by injection (e.g.. intravenous, intra ⁇ peritoneal, subcutaneous, intramuscular) .
  • a viral infection e.g.. intravenous, intra ⁇ peritoneal, subcutaneous, intramuscular
  • intravenous injection is used.
  • the mutant protein may be injected in a fluid or may be encapsulated in liposomes.
  • the mutant protein may also be applied topically to, e.g.. a wound or other site of infection.
  • the mutant proteins of the invention may also be used to treat bacterial infections, especially gram- negative bacterial infections, and endotoxic shock.
  • Endotoxins are the lipopolysaccharide components of the outer membranes of gram-negative bacteria that trigger many of the adverse systemic reactions and serious sequelae in sepsis and gram-negative bacteremia.
  • an effective amount of a mutant protein is administered to the mammal.
  • the mutant protein is preferably administered to the mammal before the bacterial infection becomes too serious and septic shock or endotoxic shock has developed.
  • the mutant protein is administered at the first indication of a bacterial infection or prophylactically to those at risk of developing bacterial infections.
  • a mutant protein may be administered prophylactically to surgical patients or patients in intensive care who are at risk of developing bacterial infections.
  • the mutant protein can be administered to a mammal already suffering from a bacterial infection or already suffering from septic shock or endotoxic shock.
  • the mutant protein will generally be administered to the mammal suffering from a bacterial infection or endotoxic shock by injection (e.g.. intravenous, intraperitoneal, subcutaneous, intramuscular) . It is preferably administered by intravenous injection.
  • the mutant protein may be administered in a fluid or may be encapsulated in liposomes.
  • the mutant protein may also be applied topically to, e.g.. a wound or other site of infection, and it should be possible to administer the mutant protein by means of a spray to treat respiratory infections.
  • mutant proteins of the invention can be used to treat cancer. It is contemplated that the mutant proteins of the invention can be used to treat a variety of cancers, including but not limited to, adenocarcinoma, lymphoma, fibrosarcoma, and leukemia, and to reduce metastasis. The mutant protein should be administered to the mammal when the presence of cancer cells in the mammal is first detected.
  • the mutant protein is preferably administered to the mammal by injection (e.g.. intravenous, intraperitoneal, subcutaneous, intramuscular) .
  • the mutant protein may be administered in a fluid or encapsulated in liposomes.
  • the dose of mutant protein that must be administered to a mammal for therapeutic treatment will vary depending on the mammal which will receive the mutant protein, the reason for administering the mutant protein (e.g.. to reduce the level of immune complexes, to treat a viral infection, to treat a bacterial infection, to treat endotoxic shock, to treat cancer) , the severity of the mammal's condition, the route of administration, and the identity of any other drugs being administered to the mammal. It is also understood that it may be necessary to give more than one dose of the mutant protein.
  • Effective dosages and schedules for administration of the mutant protein may be determined empirically, and making such determinations is within the skill of the art.
  • a dose of from about 5 ⁇ g to about 150 mg of mutant protein per kg of body weight, preferably from about 100 ⁇ g to about 20 mg per kg, will be effective for reducing the level of immune complexes, treating a viral infection, treating a bacterial infection, treating endotoxic shock or treating cancer.
  • the dose is most preferably from about 2 mg to about 10 mg per kg.
  • one dose is sufficient for treating a bacterial infection or endotoxic shock.
  • Multiple doses will generally be necessary for reducing the level of immune complexes, treating viral infections and treating cancer.
  • the interval between doses is preferably from about 1 day to about 7 days.
  • the multiple doses are most preferably administered from about 1 to about 3 days apart.
  • Administration of mutant protein should be continued until the level of immune complexes has returned to normal or until health has been restored to the mammal.
  • the mutant protein is administered to the mammal in a pharmaceutically-acceptable carrier.
  • Pharmaceutically-acceptable carriers are well known.
  • suitable carriers for the administration of the mutant proteins of the invention include fluids such as water, saline and buffers.
  • the mutant protein may also be administered encapsulated in liposomes [see Deodhar et al., Cancer Research. 42. 5084-5088 (1982); Thombre et al., Cancer Immunol. Immunother.. 16. 145-150 (1984); Barna et al., Cancer Research. 44. 305-310 (1984)].
  • the mutant proteins are preferably administered encapsulated in liposomes.
  • liposome refers to any sac-like or hollow vesicle-like structure which is capable of encapsulating a mutant protein and includes multilamellar vesicles, unilamellar vesicles, and red blood cell ghosts. Methods of preparing liposomes and encapsulating molecules in them are well known in the art.
  • the liposomes containing mutant protein are unilamellar vesicles formed by extrusion which may be prepared as described by MacDonald et al., Biochim. Biophvs. Acta. 1061:297-301 (1991).
  • the mutant protein may be incorporated into lotions, gels, cre es, etc., as is well known in the art. It is within the skill in the art to determine acceptable and optimum carriers and routes of administration.
  • the mutant proteins may be administered to the mammal alone or in combination with other drugs.
  • the mutant proteins may be administered in combination with antibiotics when used to treat a bacterial infection or may be administered with other anti-cancer agents when used for treating cancer.
  • Effective dosages and schedules for administering the mutant proteins and these other drugs together may be determined empirically as set forth above. It is expected that smaller amounts of mutant protein and anti- cancer agent (50% or less) may be employed when they are used in combination as compared to when they are used individually to treat cancer. It is also within the skill in the art to determine acceptable and optimum carriers and routes of administration for the mutant proteins and the other drugs when they are used in combination.
  • the other cancer agents which can be used in combination with the mutant proteins include cytotoxic chemotherapeutic compounds known in the art such as Thiotepa, Busulfan, Cyclophosphamide, Methotrexate, Cytarabine, Bleomycin, Cisplatin, Doxorubicin, Melphalan, Mercaptopurine, Vinblastine, and 5-Fluorouracil.
  • cytotoxic chemotherapeutic compounds known in the art such as Thiotepa, Busulfan, Cyclophosphamide, Methotrexate, Cytarabine, Bleomycin, Cisplatin, Doxorubicin, Melphalan, Mercaptopurine, Vinblastine, and 5-Fluorouracil.
  • Other suitable chemotherapeutic compounds are listed in Krakoff, CA-A Cancer Journal For Clinicians. 41:264-278 (1991) .
  • cytotoxic agents function in destroying cells and/or preventing their multiplication and are thus, useful in treating cancer.
  • Immunoadjuvants and cytokines also referred to as "biological response modifiers," are known in the art. Generally, such molecules are useful in stimulating or enhancing host defense mechanisms and are therefore useful in anti-cancer therapy. Examples of immunoadjuvants or cytokines that may be administered include interferon(s) , colony stimulating factor (CSF) , tumor necrosis factor (TNF) , hormones such as steroids, and interleukins such as IL-1, IL-2, and IL-6.
  • CSF colony stimulating factor
  • TNF tumor necrosis factor
  • steroids interleukins
  • interleukins such as IL-1, IL-2, and IL-6.
  • the mutant proteins may also be used as imaging agents for detecting immune complexes or cancer cells in vivo.
  • a labeled mutant protein will be injected into a mammal and will localize in areas where immune complexes have been deposited (such as areas of inflammation) or in areas where cancer cells are located.
  • an unlabeled mutant protein can be injected.
  • a labeled material which binds to the mutant protein is injected, and this labeled material binds to the mutant protein in areas where it has localized.
  • mutant proteins of the invention can be prepared by expression of DNA coding for them in transformed host cells.
  • DNA coding for a mutant protein according to the invention can be prepared by in vitro mutagenesis of a CRP genomic or cDNA clone or can be chemically synthesized.
  • probes can readily be prepared so that genomic and cDNA clones can be isolated which code for CRP's from other species.
  • Methods of preparing such probes and isolating genomic and cDNA clones are well known. See, e.g.. Lei et al.. J. Biol. Chem.. 260. 13377-83 (1985); Woo et al., J. Biol. Chem.. 260, 13384- 88 (1985); Hu et al., Biochem.. 15, 7834-39 (1986); Hu et al., J. Biol. Chem.. 263. 1500-1504 (1988); Whitehead et al., Biochem. J.. 266. 283-90 (1990).
  • DNA coding for a mutant protein according to the invention can be prepared using conventional and well known in vitro mutagenesis techniques. Particularly preferred is site-directed mutagenesis using polymerase chain reaction (PCR) amplification. See Example l.
  • PCR polymerase chain reaction
  • the following references describe other site-directed mutagenesis techniques which can be used to produce DNA coding for a mutant protein of the invention: Current Protocols In Molecular Biology. Chapter 8, (Ansubel ed. 1987) ; Smith & Gilliam, Genetic Engineering Principles And Methods. 3 , 1-32 (1981); Zoller & Smith, Nucleic Acids Res..
  • DNA coding for a mutant protein of the invention can also be prepared by chemical synthesis.
  • Methods of chemically synthesizing DNA having a specific sequence are well-known in the art. Such procedures in ⁇ clude the phosphoramidite method (see, e.g.. Beaucage and Caruthers, Tetrahedron Letters. 22. 1859 (1981); Matteucci and Caruthers, Tetrahedron Letters. 21. 719 (1980) ; and Matteucci and Caruthers, J. Amer. Chem. Soc.. 103. 3185 (1981)) and the phosphotriester approach (see, e.g.. Ito et al., Nucleic Acids Res.. 10. 1755-69 (1982)) .
  • the invention also includes a vector which comprises a DNA sequence coding for a mutant protein according to the invention.
  • the DNA coding sequence is operatively linked in the vector to appropriate expression control sequences. Methods of effecting this operative linking, either before or after the DNA coding for the mutant protein is inserted into the vector, are well known.
  • Expression control sequences include pro ⁇ moters, activators, enhancers, operators, stop signals, cap signals, polyadenylation signals, and other signals involved with the control of transcription.
  • the vector must contain a promoter and a trans ⁇ cription termination signal, both operatively linked to the DNA sequence, i.e.. the promoter is upstream of the DNA sequence and the termination signal is downstream from it.
  • the promoter may be any DNA sequence that shows transcriptional activity in the host cell and may be derived from genes encoding homologous or heterologous proteins and either extracellular or intracellular proteins, such as amylase, glycoamylases, proteases, lipases, cellulases, and glycolytic enzymes.
  • a promoter recognized by T7 RNA polymerase may be used if the host is also engineered to contain the gene coding for T7 RNA polymerase.
  • the promoter may contain upstream or downstream activator and enhancer sequences. An operator sequence may also be included downstream of the promoter, if desired.
  • the promoter need not be identical to any naturally-occurring promoter. It may be composed of portions of various promoters or may be partially or totally synthetic. Guidance for the design of promoters is provided by studies of promoter structure such as that of Harley and Reynolds, Nucleic Acids Res.. 15. 2343-61 (1987) . Also, the location of the promoter relative to the transcription start may be optimized. See Roberts, et al., Proc. Natl Acad. Sci. USA. 76. 760-4 (1979).
  • Expression control sequences suitable for use in the invention are well known. They include those of the E. coli lac system, the E. coli trp system, the TAC system and the TRC system; the major operator and pro ⁇ moter regions of bacteriophage lambda; the control region of filamentous single-stranded DNA phages; the expression control sequences of other bacteria; promoters derived from genes coding for Saccharomyces cerevisiae TPI, ADH, PGK and alpha-factor; promoters derived from genes coding for the Aspergillus oryzae TAKA amylase and A.
  • niger glycoamylase niger glycoamylase, neutral alpha-amylase and acid stable alpha-amylase
  • promoters derived from genes coding for Rhizomucor miehei aspartic proteinase and lipase mouse mammary tumor promoter; SV40 promoter; the actin pro ⁇ moter; and other sequences known to control the expression of genes of prokaryotic cells, eukaryotic cells, their viruses, or combinations thereof.
  • the vector may be a self-replicating vector or an integrative vector. If the vector is self- replicating, it must contain one or more replication systems which allow it to replicate in the host cells. In particular, when the host is a yeast, the vector should contain the yeast 2u replication genes REP1-3 and origin of replication.
  • the vector When the vector is a self-replicating vector, it is preferably a high copy number plasmid so that high levels of expression are obtained.
  • a "high copy number plasmid" is one which is present at about 100 copies or more per cell.
  • Many suitable high copy number plasmids are known and include bacterial plasmids such as pUC and yeast plasmids such as pC.
  • an integrating vector may be used which allows the integration into the host cell's chromosome of the DNA coding for the mutant proteins.
  • the copy number of the coding sequences in the host cells would be lower than when self-replicating vectors are used, transformants having sequences integrated into their chromosomes are generally quite stable.
  • the vector should further include one or more restriction enzyme sites for inserting DNA sequences into the vector, and preferably contains a DNA sequence coding for a selectable or identifiable phenotypic trait which is manifested when the vector is present in the host cell ("a selection marker") .
  • a selection marker a DNA sequence coding for a selectable or identifiable phenotypic trait which is manifested when the vector is present in the host cell.
  • Suitable vectors for use in the invention are well known. They include retroviral vectors, vaccinia vectors, pUC (such as pUC8 and pUC4K) , pBR (such as pBR322 and pBR328) , pTZ (such as pTZ18R) , pUR (such as pUR288) , phage lambda, YEp (such as YEp24) plasmids, and derivatives of these vectors.
  • retroviral vectors such as pUC8 and pUC4K
  • pBR such as pBR322 and pBR328
  • pTZ such as pTZ18R
  • pUR such as pUR288
  • phage lambda phage lambda
  • YEp such as YEp24
  • DNA coding for a signal or signal-leader sequence may be located upstream of the DNA sequences encoding the mutant proteins.
  • a signal or signal-leader sequence is an amino acid sequence at the amino terminus of a protein which allows the protein to which it is attached to be secreted from the cell in which it is produced. Suitable signal and signal-leader sequences are well known and include the CRP presequence (see Background section) , yeast «-factor signal sequence (see U.S. Patent Nos. 4,546,082 and 4,870,008), the yeast BARl secretion system (see U.S. Patent No.
  • Chemical synthesis of DNA coding and other sequences is preferable for several reasons. First, chemical synthesis is desirable because codons preferred by the host in which the DNA sequence will be expressed may be used to optimize expression of the mutant proteins. Not all of the codons need to be altered to obtain improved expression, but greater than 50%, most preferably at least about 80%, of the codons should be changed to host-preferred codons.
  • the codon preferences of many host cells including E. coli. yeast, and other prokaryotes and eukaryotes, are known. See Maximizing Gene Expression, pages 225-85 (Reznikoff & Gold, eds., 1986).
  • the codon preferences of other host cells can be deduced by methods known in the art. In particular, the following method is generally used. First, the codon usage in genes coding for about a dozen proteins which are highly expressed in the intended host cell is determined. Then, the codon usage in genes coding for about a dozen proteins expressed at low levels in the host cell is determined. By reviewing the results, codons that are used often and those that are used rarely are identified. The codons that are used most often in highly-expressed genes are preferred. Those used rarely in highly-expressed genes or used in genes expressed at low levels are preferably not used.
  • Chemical synthesis also allows for the use of optimized expression control sequences with the DNA sequences coding for the mutant proteins. In this manner, optimal expression of the mutant proteins can be obtained.
  • promoters can be chemically synthesized and their location relative to the transcription start optimized.
  • an optimized ribosome binding site and spacer can be chemically synthesized and used with coding sequences that are to be expressed in prokaryotes.
  • the site at which the ribosome binds to the messenger includes a sequence of 3- 9 purines.
  • the consensus sequence of this stretch is 5'- AGGAGG-3' , and it is frequently referred to as the Shine- Dalgarno sequence.
  • the sequence of the ribosome binding site may be modified to alter expression. See Hui and DeBoer, Proc. Natl. Acad. Sci. USA. 84., 4762-66 (1987). Comparative studies of ribosomal binding sites, such as the study of Scherer, et al., Nucleic Acids Res.. 8_, 3895-3907 (1987), may provide guidance as to suitable base changes.
  • the ribosome binding site lies 3-12 bases upstream of the start (AUG) codon.
  • a ribosome binding site and spacer that provide for efficient translation in the prokaryotic host cell should be provided.
  • a preferred ribosome binding site and spacer sequence for optimal translation in E. coli are described in Springer and Sligar, Proc. Nat'l Acad. Sci. USA. 84. 8961-65 (1987) and von Bodman et al., Proc. Nat'l Acad. Sci. USA. 8_3, 9443-47 (1986) .
  • the consensus sequence for the translation start sequence of eukaryotes has been defined by Kozak (Cell. 44, 283-292 (1986)) to be: C(A/G)CCAUGG. Deviations from this sequence, particularly at the -3 position (A or G) , have a large effect on translation of a particular mRNA. Virtually all highly expressed mammalian genes use this sequence. Highly expressed yeast mRNAs, on the other hand, differ from this sequence and instead use the sequence (A/Y)A(A/U)AAUGUCU (Cigan and Donahue, Gene. 59. 1-18 (1987)). These sequences may be altered empirically to determine the optimal sequence for use in a particular host cell.
  • a host cell capable of expressing a mutant protein according to the invention can be prepared by transforming the cell with a vector comprising a DNA sequence that codes for the mutant protein.
  • a DNA molecule coding for a mutant protein may be used to transform the host cell. Methods of transforming cells with vectors or DNA are well known in the art.
  • Any of a large number of available and well-known host cells may be used in the practice of this invention.
  • the selection of a particular host is de ⁇ pendent upon a number of factors recognized by the art. These include, for example, compatibility with the chosen expression vector, toxicity to it of the mutant protein encoded for by the DNA sequence, rate of transformation, expression characteristics, bio-safety and costs. A balance of these factors must be struck with the understanding that not all hosts may be equally effective for the expression of a particular mutant protein.
  • useful hosts include bacteria (such as E. coli sp.), yeast (such as Saccharomyces sp.) and other fungi, insects, plants, animals (including human), or other hosts known in the art.
  • mutant proteins of the invention may be produced by culturing the chosen host cell under conditions which permit expression of the mutant protein.
  • Methods of culture and culture media are well known in the art, but the use of enriched media (rather than minimal media) is preferred since much higher yields are obtained.
  • Restriction enzymes used in the following examples were obtained from various commercial sources and used according to the manufacturer's instructions or by employing a standard buffer system (Maniatis et al., Molecular Cloning: A Laboratory Manual (1982)).
  • This example describes the preparation of a recombinant DNA molecule coding for a mutant human CRP subunit in which the two cysteine residues at positions
  • Bold bases indicate mutagenized codons and the initiator ATG codon.
  • Underlined bases are complementary to the CRP cDNA sequence.
  • the five reactions were: (1) Reaction of cDNA clone coding for preCRP with primers 1 and 2 to produce PCR product A; (2) Reaction of cDNA coding for preCRP with primers 3 and 4 to produce PCR product B; (3) Reaction of cDNA coding for preCRP with primers 5 and 6 to produce PCR product C; (4) Reaction of products A and B in the presence of primers 1 and 4 to produce PCR product DI; and (5) Reaction of products DI and C in the presence of primers 1 and 6 to produce the final product D2.
  • D2 was thought to code for the mature sequence of human CRP subunit (the presequence has been eliminated) , except that there was an additional methionine at the N-terminus and cysteines 36 and 97 had been replaced by alanines. This was subsequently found not to be the case (see section F below) .
  • the DNA coding for human preCRP used as the starting material for these PCR reactions was obtained by digestion of pCRP5 with iS ⁇ oRI to yield linear (noncircular) DNA.
  • a sample of plasmid pCRP5 was obtained from Drs. Bruce Dowton and Harvey Colten of Washington University School of Medicine, St. Louis, MO.
  • pCRP5 was isolated from a human liver cDNA library as described in Tucci et al., J. Immunol.. 131. 2416-19 (1983) .
  • the nucleotide sequence of the cDNA of pCRP5 and the amino acid sequence of the preCRP coded for by it are given in Woo, et al., J. Biol. Chem.. 260. 13384-13388 (1985) .
  • the cDNA of the pCRP5 sample obtained from Drs. Dowton and Colten was found to have a deletion in codon 48.
  • PCR reactions were carried out using VENT polymerase (New England Biolabs) to minimize unwanted mutations due to misincorporation of bases, and the PCR reactions were done using 20 cycles, each cycle consisting of: 94°C for 1 minute; 37°C, 42°C or 60°C for 1 minute (the annealing temperature depending on the sequence of the primers) ; and 74°C for 3 minutes. Following the amplification steps, the reactants were further incubated at 74°C for 5 minutes to complete the synthesis of double-stranded DNA. Each of the products was purified by agarose gel electrophoresis as described in Horton et al., supra. For the PCR reactions where the template consisted of two overlapping sequences (PCR DI and PCR D2 in Figure IA) , the reactants were incubated without primers for 4 cycles to allow the formation of full-length template before normal amplification was carried out.
  • VENT polymerase New England Biolabs
  • the final PCR product D2 was concentrated by filtration through a Centricon 30 apparatus (Amicon, Beverly, MA) , and then treated with T4 polynucleotide kinase (Pharmacia, Piscataway, NJ) and T4 DNA ligase (New England Biolabs, Inc.) as described in Denney et al., Amplifications. 4_, 25-26 (1990) .
  • the resultant material was digested with Ndel and Bgrlll to release the mutant CRP coding sequence, and the released coding sequence was ligated to the expression vector pETV which had been digested with Ndel and BamHI and treated with calf intestinal alkaline phosphatase (Promega, Madison, WI) .
  • the ligation mixture was used to transform E. coli DH5 ⁇ (Gibco BRL Life Technologies, Inc.), and transformants were screened by inipreps performed as described in Birnboim et al., Nucleic Acids Res.. 1, 1513-1523 (1979) to identify the correct plasmid pIT4.
  • a restriction map of pIT4 is provided in Figure IB. As shown in Figure IB, the mutant CRP coding sequence is under the control of the T7 promoter.
  • Plasmid pETV is a derivative of pET3a.
  • the preparation of pET3a is described in Rosenberg et al., Gene, 56. 125-135 (1987) .
  • Plasmid pET3a was obtained from Dr. W. Studier, Brookhaven National Laboratory, Upton NY.
  • Plasmid pET3a has two Nhel restriction enzyme sites. One is located at the T7 gene 10 translation start site in which it was desired to insert the mutant CRP coding sequence. The second Nhel site is located within a 190-bp fragment bounded by EcoRV sites. This second Nhel site was eliminated by digestion with JScoRV and recircularizing the plasmid to yield pETV.
  • Plasmid pIT4 was digested with XJal and Kpnl , and the relevant fragment subcloned into M13mpl8RF (Yanisch-Perron et al., Gene. 3_3, 103-119 (1985)). Single-stranded DNA was obtained from cultures carrying M13mpl8RF and sequenced as described by Sanger et al., J. Mol. Biol.. 94. 441-558 (1975) using an Applied Biosystems Model 370A Automated DNA Sequencer.
  • Plasmid pIT4 was used to transform E. coli BL21(DE3) (preparation described in Studier et al., J. Mol. Biol.. 189. 113-130 (1986)) which carries the phage T7 RNA polymerase gene under expression control of the lacUV5 operator and promoter. Competent E. coli BL21(DE3) was obtained from Novogen, Madison, WI. Transformants were selected on LB medium (Miller, Experiments In Molecular Genetics (1972) ) containing 50 ⁇ g/ml ampicillin.
  • Transformed cells were grown in M9ZB medium (Studier et al., J. Mol. Biol.. 189. 113-130 (1986)) containing 100 ⁇ g/ml ampicillin at 37°C for small-scale experiments.
  • Ten-liter cultures were grown in a New Brunswick Microgen SF-116 fermentor in 2YT medium (Miller, Experiments In Molecular Genetics (1972)) plus 0.4% (w/v) glucose and lOO ⁇ g/ml ampicillin at 37°C with aeration using compressed air at a rate of 10 liters per minute.
  • the harvested cells were suspended in 20mM Tris-HCl, pH 7.5, containing 5mM EDTA (40 g in 500 ml) and disrupted by three passages through a Manton-Gaulin homogenizer at 10,000 psi.
  • the extract was centrifuged in a Beckman JA10 rotor at 8,000 rpm for 20 min. at 2°C.
  • the pellet containing the insoluble mutant CRP subunits was washed twice with 20 ml of the same buffer containing 0.5% (v/v) Triton X-100 (Bio-Rad) , followed by centrifugation in a JA18 rotor at 9,000 rpm for 20 min. to remove the soluble material.
  • mutant rCRP inclusion body preparation 10 mM Tris-HCl, pH 7.5-8.0, and incubated at 4°C overnight with mixing to solubilize the mutant CRP subunits (this material is referred to hereinafter as "mutant rCRP inclusion body preparation") .
  • this material was diluted with lOmM Tris-HCl, pH 7.5-8.0, to a final concentration of 6M urea and then loaded onto a column containing 40 cc of Q- Sepharose Fast Flow 1 * anion exchange resin (Pharmacia) at 6 ml per minute.
  • Bound materials were eluted with a linear NaCl gradient using lOmM Tris-HCl, pH 7.5-8.0, containing 6M urea and 1 M NaCl. Absorbance at 280 nm was measured using a BioPilot R automated chromatography system (Pharmacia) , and an elution profile was obtained.
  • native CRP, mCRP and the primary translation product of a CRP cDNA clone were also chromatographed on Q-Sepharose Fast Flow 1 *.
  • Native CRP, mCRP and wild- type rCRP were prepared and chromatographed as follows.
  • Native CRP was isolated from pleural or ascites fluid by calcium-dependent affinity chromatography using phosphorylcholine-substituted BioGel R A 0.5m (an agarose- based resin obtained from BioRad Laboratories) as described by Volanakis, et al. [J. Immunol.. 113. 9-17 (1978)] and modified by Potempa, et al. [Mol. Immunol.. 24. 531-41 (1987)]. Briefly, the pleural or ascites fluid was passed over the phosphorylcholine-substituted column, and the CRP was allowed to bind.
  • the column was exhaustively washed with 75 mM Tris-HCl- buffered saline (pH 7.2) containing 2 mM CaCl 2 until the absorbance at 280 nm was less than 0.02.
  • the CRP was eluted with 75 mM Tris, 7.5 mM citrate-buffered saline (pH 7.2). This high concentration of Tris significantly reduces non-specifically adsorbed proteins which often contaminate affinity-purified CRP preparations.
  • CRP-containing fractions were pooled, diluted three-to-five fold with deionized water, adsorbed to Q-Sepharose Fast Flow 1 * ion exchange resin, and then eluted with a linear salt gradient of from 0-1 M NaCl in lOmM Tris-HCl, pH 7.4.
  • CRP-containing fractions were pooled and re- calcified to 2-5mM CaCl 2 (by adding a suitable amount of a 1M solution) and applied to unsubstituted Biogel 1 * A 0.5m column to remove residual serum amyloid P component (SAP) .
  • the CRP was concentrated to l mg/ml using ultrafiltration (Amicon; PM30 membrane) under 10-20 psi nitrogen. A CRP extinction coefficient (mg/ml) of 1.98 was used to determine concentration.
  • the concentrated CRP was exhaustively dialyzed against 10 mM Tris-HCl-buffered saline, pH 7.2, containing 2mM CaCl 2 . This preparation produced a single Mr 23,000 band on SDS- PAGE electrophoresis and was more than 99% free of SAP, IgG and all other proteins tested for antigenically.
  • Bound material was eluted with a linear NaCl gradient using lOmM Tris-HCl, pH 7.5-8.0, containing 6M urea and 1 M NaCl.
  • a 280 was measured using the BioPilot R automated chromatography system.
  • the wild-type rCRP was prepared by expression of pIT3 in E. coli BL21(DE3).
  • Plasmid pIT3 was prepared by cleaving pCRP5 with MscI and Bglll (see Figure 2A) .
  • pETV was cleaved with Nhel and BamHI, and the digested pETV and pCRP5 were mixed and ligated (see Figure 2A) .
  • This ligation mixture was used to transform E. coli strain DH5 ⁇ and colonies carrying the desired expression plasmid pIT3 were identified, all as described above for pIT4.
  • Plasmid pIT3 was thought to code for a CRP subunit having the sequence of the unmutated human CRP subunit, except that it has the peptide Met Ala Ser at the N-terminus (see Figure 2B) . However, this was subsequently found not to be the case (see section F below).
  • E. coli BL21(DE3) were transformed with pIT3 and cultured as described above for pIT4. The cultures were induced, the cells were harvested, and a pellet containing the wild-type rCRP was obtained, all as described above.
  • Bound materials were eluted with a linear NaCl gradient using lOmM Tris-HCl, pH 8.0, containing 6M urea and 1 M NaCl.
  • a 280 was measured using the BioPilot R system.
  • the elution profiles generated by the BioPilot R system for native CRP, mCRP, wild-type rCRP and the mutant CRP subunits are shown in Figures 3A-D.
  • the BioPilot R system was programmed so that the salt gradient used in each case could be directly compared.
  • native CRP gave only one significant elution peak. It had an elution time of 47.76 minutes. This peak was dialyzed against 25mM Tris- HCl, 0.15M NaCl, 2 mM CaCl 2 , pH 7.4, and then concentrated to about 1 mg/ml using Amicon filtration for testing as described in the next section.
  • the concentrated peak material is referred to hereinafter as "native-CRP Q ".
  • Figure 3B shows the elution profile for mCRP.
  • the predominant peak has an elution time of 36.18 minutes.
  • This peak was dialyzed against low ionic strength buffer (25 mM Tris-HCl, 0.015 M NaCl, pH 7.4) and then concentrated using Amicon filtration and was tested as described in the next section.
  • the concentrated peak material is referred to hereinafter as "mCRP ⁇ j ".
  • the testing described in the next section verified that the material in the peak was mCRP.
  • mCRP elutes from Q-Sepharose R much earlier (approximately 11.5 minutes earlier) than native CRP.
  • Figure 3C shows the elution profile for wild- type rCRP.
  • the predominant peak eluted at 34.55 minutes, an elution time almost identical to that of mCRP and very distinct from the elution time of native CRP.
  • the protein in the predominant peak was dialyzed against low ionic strength buffer and then concentrated using Amicon filtration for further testing as described in the next section.
  • the concentrated peak material is referred to hereinafter as "wild-type rCRP Q ".
  • Figure 3D shows the elution profile for the mutant CRP subunits.
  • the main peak eluted at 34.55 minutes, an elution time essentially identical to that of wild-type rCRP and mCRP, and distinct from that of native CRP.
  • the main peak was dialyzed against low ionic strength buffer and then concentrated using Amicon filtration for further testing as described in the next section.
  • the concentrated peak material is referred to hereinafter as "mutant rCRP Q ".
  • Proteins may also be eluted from the Q- Sepharose Fast Flow 1 * column with an NaCl step gradient (from 0.1 to 1.0 M NaCl).
  • an NaCl step gradient from 0.1 to 1.0 M NaCl.
  • the A 280 readings showed that most of the protein was eluted from the column with 0.1 - 0.2 M NaCl.
  • native CRP was run on the column and eluted with a step gradient
  • the A 280 readings showed that most of the protein eluted with 0.4 M NaCl.
  • Blocking solution 100 ⁇ l of 1% BSA in TBS was added to the wells and incubated for 30 min. at room temperature (RT) , and vacuum-filtered through the membrane. The wells were washed three times with TBS containing 1% BSA and 0.05% Tween 20 (TBS washing buffer). Mouse monoclonal antibody (mAb) 3H12 and antiserum LP3-HRP were added and incubated for 30 min. at RT followed by washing.
  • mAb monoclonal antibody
  • Monoclonal antibody 3H12 was used unlabeled, and the blots were developed by adding rabbit anti-mouse IgG F(ab') 2 labeled with horseradish peroxidase (Southern Biotechnology Associates, Birmingham, AL) , incubating for 30 min. at RT, adding peroxidase substrate 4-chloro-2- naphthol (Bio-Rad) in 10 mM Tris-HCl, 0.15 M NaCl, containing methanol and H 2 0 2 prepared as directed (Bio ⁇ Rad) , and incubating for 30 min. at RT to allow for color development.
  • LP3-HRP was labeled with horseradish peroxidase, and the blots were developed by adding 4- chloro-2-naphthol followed by an incubation for 30 min. at RT for color development.
  • Monoclonal antibody 3H12 is an IgG antibody specific for an antigenic determinant found on mCRP but not on native CRP. Its preparation and properties are described in U.S. Patent No. 5,272,257, PCT application WO 91/00872, Ying et al., J. Immunol.. 143. 221-228 (1989) and Ying et al., Mol. Immunol.. 19, 677-87 (1992).
  • Antiserum LP3 is an antiserum prepared by immunizing a goat with mCRP in complete Freund's adjuvant and then affinity-purifying the harvested antiserum by passing it over a column of cyanogen bromide-activated BioGel R substituted with mCRP. The resulting affinity-purified anti-neoCRP antiserum LP3 was monospecific for the neo- CRP antigenicity expressed by mCRP but not by native CRP. LP3 was labelled with horseradish peroxidase as described in Potempa et al., Molec. Immunol.. 24. 531-541 (1987).
  • the peaks were also analyzed by Western blot. To perform the Western blot, 5-10 ⁇ l of the peak concentrates were electrophoresed on 12% SDS-PAGE gels under reducing and non-reducing conditions. After electrophoresis, protein was transferred to a nitrocellulose membrane using the JKA Biotech (Denmark) Semidry Electroblotter. The remainder of the procedure was the same as described above for the dot blot, except that three mouse mAbs (3H12, 2C10 and 8C10) were used. The color was developed as described in the previous section for 3H12.
  • Monoclonal antibody 3H12 is described above.
  • Monoclonal antibody 8C10 reacts with a determinant found only on mCRP, whereas 2C10 reacts with a determinant found on both native CRP and mCRP.
  • the preparation and properties of 2C10 and 8C10 are described in U.S. Patent No. 5,272,257, PCT application WO 91/00872, Ying et al., J. Immunol.. 143. 221-228 (1989) and Ying et al., Mol. Immunol.. 29. 677-87 (1992).
  • Lane 2 contains the mutant rCRP inclusion body preparation. Note that there are two predominant bands, including one at approximately the same position as the single mCRP band (Mr of about 27,000). This band was verified by Western blot analysis to be antigenically reactive with antibodies specific for mCRP determinants.
  • Lane 3 contains mutant rCRP Q (the mutant rCRP inclusion body preparation which had been chromatographed on Q-Sepharose Fast Flow 1 *) . Note that only one major band was obtained having a molecular weight of approximately 27,000, the molecular weight of the desired free mutant CRP subunits. As can be seen, there are many fewer bands as compared to the mutant rCRP inclusion body preparation (lane 2) , and the amounts of remaining contaminants, especially the one predominant contaminant having Mr less than 18,000, have been substantially reduced. Thus, the purity of the mutant CRP subunit was greatly improved by the single-step Q-Sepharose Fast Flow R chromatography procedure.
  • Lane 4 contains wild-type rCRP inclusion body preparation. As can be seen, multiple protein bands are present. The band approximately 80% down the lane is believed to be the free subunit (Mr of about 27,000). Note that it is not the predominant band, with the band at the bottom of the lane being of greater intensity.
  • Lane 5 contains wild-type rCRP Q (the wild-type rCRP inclusion body preparation which had been chromatographed on Q-Sepharose Fast Flow 1 *) .
  • wild-type rCRP Q the wild-type rCRP inclusion body preparation which had been chromatographed on Q-Sepharose Fast Flow 1 *
  • multiple bands are still present as compared to the unchromatographed inclusion body preparation (lane 4) . Indeed, some new bands have appeared (at approximate molecular weights of 45,000 and 14,000), the identity of which is unknown, and the intensity of some bands has increased. Thus, no improvement in purification of wild- type rCRP was achieved using the Q-Sepharose Fast Flow 1 * column.
  • the band about 80% down the lane is believed to be the free subunit. This band and many of the other bands were reactive with the antibodies specific for mCRP determinants by Western blot analysis. This indicates that most of the protein recovered after chromatography of wild-type rCRP preparations
  • Lane 6 contains Prestained BioGel R molecular weight standards (BioRad) . From top to bottom, these bands are of approximate molecular weight 106,000, 80,000, 49,500, 32,500, 27,500 and 18,500.
  • ELISA was performed to detect binding of antibodies specific for native CRP and mCRP to the materials in the peaks eluted from the Q-Sepharose Fast Flow R columns.
  • a direct binding ELISA was used for mCRP and recombinant CRP preparations.
  • a ligand capture ELISA was used for native CRP preparations.
  • test protein 5 ⁇ g/ml in 50mM sodium bicarbonate buffer (pH 9.5) were placed in the wells of Nunc polystyrene plates (Scientific Supply, Shiller Park, IL) and incubated for 2 hr at 37°C or 4°C overnight.
  • TBS 25mM Tris-HCl, 0.15M NaCl, pH 7.4
  • BSA bovine serum albumin
  • TBS-A bovine serum albumin
  • Antibodies were serially diluted with TBS-A, and 100 ⁇ l aliquots were added to the wells and incubated for 60 min. at 37°C, followed by washing. Peroxidase-conjugated rabbit anti-mouse IgG (Southern Biotech) in TBS-A was added to the wells for 60 min. at 37°C. After washing, 100 ⁇ l ABTS substrate (2- 2'azino-bis(3-ethylbenzylthiazoline-6-sulfonic acid, Sigma Chemical Co.) were added per well and incubated for about 5-15 min. at RT. Plates were read at an absorbance of 414 nm on a Titertek R multiskan plate reader (Flow Laboratories, Helsinki, Finland) .
  • PC-KLH is phosphorylcholine (PC) substituted Keyhole Limpet hemocyanin (KLH) .
  • Figures 5A-D show the results of these ELISAs.
  • Figure 5A shows the reactivity of mCRP, wild-type rCRP inclusion body preparation and native CRP with mAb 3H12. As expected mAb 3H12 reacted with mCRP but not with native CRP. Also, wild-type rCRP inclusion body preparation reacted like mCRP and unlike native CRP, indicating that the mCRP epitope recognized by mAb 3H12 (the carboxy-terminal octapeptide of the CRP subunit) is expressed on wild-type rCRP.
  • Figure 5B shows the reactivity of mCRP, wild- type rCRP inclusion body preparation and native CRP with mAb 1D6.
  • Monoclonal antibody 1D6 is specific for an antigenic determinant found only on native CRP. Its preparation and properties are described in U.S. Patent No. 5,272,257, PCT application WO 91/00872, Ying et al., J. Immunol.. 143. 221-228 (1989) and Ying et al., Mol. Immunol.. 29. 677-87 (1992).
  • mAb 1D6 reacted with native CRP but not with mCRP.
  • wild-type rCRP inclusion body preparation like mCRP, did not react with mAb 1D6, indicating that the native CRP epitope recognized by mAb 1D6 is not expressed on wild-type rCRP.
  • Figure 5C shows the reactivity of mCRP, wild- type rCRP Q and mutant rCRP ⁇ with mAb 8C10.
  • Monoclonal antibody 8C10 reacts with an epitope found only on mCRP, but reacts with a different epitope on mCRP than does mAb 3H12.
  • each test protein was adjusted so that 1000 ng of protein was immobilized on the first well of the polystyrene ELISA plate. The test proteins were then serially diluted so that less and less protein was immobilized per well. The amount of protein that was immobilized per well is indicated on the abscissa of the graph in Figure 5C.
  • Figure 5D shows the reactivity of mCRP, wild- type rCRP ⁇ and mutant rCRP Q with affinity-purified antiserum LP3-HRP specific for neo-CRP antigenicity.
  • each test protein was adjusted so that 1000 ng of protein was immobilized on the first well of the polystyrene ELISA plate. The test proteins were then serially diluted so that less and less protein was immobilized per well. The amount of protein that was immobilized per well is indicated on the abscissa of the graph in Figure 5D.
  • an ELISA was performed to detect binding to aggregated IgG of the material in the peaks from the Q-Sepharose Fast Flow 1 * columns. Binding to aggregated immunoglobulins and immune complexes is an important activity of mCRP which permits it to be used to removed aggregated immunoglobulins and immune complexes from fluids and to quantitate immune complexes. This ELISA was performed as follows.
  • test protein 10 ⁇ g/ml
  • lOmM sodium bicarbonate buffer pH 9.5
  • TBS wash buffer 100 ⁇ l of aggregated human IgG or monomeric IgG at varying concentrations in TBS-A were added and incubated for 60 min. at 37°C.
  • Human immune globulin (U.S.P.-Gammar R , Armour Pharmaceutical Co.) was diluted to 20 mg/ml in buffer at pH 9.0 and was aggregated by heating to 63°C for about 30 minutes.
  • Non-aggregated monomeric IgG was separated from the aggregated IgG by molecular sieve chromatography using BioGel R A 1.5m in 25 mM Tris-HCl, 0.3M NaCl, pH 7.4 at 4°C.
  • LP4-HRP monospecific goat anti-neo-CRP antiserum labeled with horseradish peroxidase
  • N-terminal sequence analysis of the purified fragment confirmed that the fragment contained the expected CRP sequence from residues 1 to 6.
  • the N-terminal sequence analysis was performed by Analytical Biotechnology
  • the Mr 7000 fragment used for the N-terminal sequencing was purified as follows.
  • the concentration of protein in an inclusion body suspension was estimated using the Bicinchoninic Acid protein assay (Pierce Biochemicals) .
  • Inclusion body protein was then solubilized in 6 M guanidine HCl (GuHCl; J.T. Baker Inc.), 15 mM Tris-Cl, pH 7.2, 10 mM EDTA at a concentration of 4 mg protein/ ml.
  • the column was then developed with a four-column volume gradient of 0-0.25 M NaCl in the equilibration buffer (6M urea, 25 mM sodium acetate, pH 4.0). The fragment eluted at approximately 0.24 M NaCl. Fractions containing the fragment were pooled and concentrated by precipitation with 50% saturated ammonium sulfate. The precipitate was collected by centrifugation as described above and dissolved in equilibration buffer. The dissolved precipitate was again passed over an S- Sepharose Fast Flow column as described above, except that elution was performed with a 0-0.8 M NaCl gradient.
  • the DNA coding for the mutant rCRP was sequenced. To do so, the coding sequence was excised from pIT4 using Xbal and Kpnl and ligated into pBluescript KS (Stratagene Inc.) previously digested with the same enzymes. The subcloned DNA was sequenced on an Applied Biosystems Sequenator by the dideoxy chain termination method using standard M13-based primers. The DNA sequences of two independent isolates revealed two differences with respect to the published Woo et al. sequence (Woo et al., J. Biol. Chem.. 260. 13384-13388 (1985)).
  • C-terminal sequencing of the purified fragment was performed by Analytical Biotechnology Services. Briefly, the fragment was digested with carboxypeptidase Y at a ratio of 5 ⁇ g enzyme per 10 n ol protein (based on an estimated molecular weight of 7000) . The mixture was incubated at 37°C, and 0.5 nmol aliquots were removed at 0, 30, 60 and 180 minutes and frozen at -20°C. Each aliquot was then subjected to amino acid analysis using the Waters PICO-TAG system.
  • T at codon 47 would also fortuitously introduce a novel restriction site into the DNA by changing the base sequence from CCCGTGG to CCCGGG, the latter being the recognition sequence for the enzyme Smal .
  • Analytical digests were therefore performed on pIT3, pIT4 and pCRP5 by digesting 50-100 ng of each plasmid for 2-4 hours at 37°C with EcoRI , Smal , Kpnl or Smal and Kpnl. The digestion products were separated on 1% agarose gels and visualized with ethidium bromide. All three plasmids were digested with Smal yielding fragments of the sizes expected if the T in codon 47 were missing. These results further confirm the results of the DNA sequence analysis. These results also demonstrate that the deletion had occurred in the original plasmid pCRP5 and was not a result of the subcloning process described in section A above. No undigested DNA was detectable in any of the plasmid digests.
  • This segment differs greatly in amino acid composition from the known CRP sequence, so amino acid analyses were performed on the purified wild-type and mutant rCRP produced by pIT3 and pIT4. These proteins were purified as follows. Mutant rCRP was purified using a two-step column procedure. Inclusion body protein was solubilized, fractionated with ammonium sulfate and dissolved in urea as described above for the Mr 7000 fragment. This material was then applied to an S- Sepharose Fast Flow cation exchange column. The column was developed with a 0-0.25 M NaCl gradient in 6M urea, 25mM sodium acetate, pH 4.0, to removed impurities.
  • the mutant rCRP was eluted with 8M GuHCl, 25 mM sodium acetate, pH 4.0.
  • the GuHCl eluate was then applied to a Superdex 200 gel filtration column (Pharmacia) which had been equilibrated with 3 M GuHCl, 4 M urea in 25 mM sodium acetate, pH 4.0.
  • the wild-type rCRP was purified using a similar procedure, except that the S-Sepharose Fast Flow column was omitted and the mobile phase for the Superdex-200 gel filtration chromatography was 25 mM Tris-Cl, pH 8.0, containing 6 M GuHCl and 2 mM dithiothreitol (Pierce Biochemicals) .
  • the purified proteins were submitted to Analytical Biotechnology Services for complete amino acid composition analysis. The results (presented in Tables 4a and 4b below) were inconclusive.
  • Nr 7000 Possible amino acid sequence of Nr 7000 fragment between residues 46-72 assuming deletion only and corresponding DNA sequence:
  • SEQ ID NO:18 o The nucleotides deleted or inserted are underlined when present. Amino acids in boldface type are those differing from the sequence of CRP.
  • the base deletion in codon 47 of the CRP coding sequence of pIT4 was preventing efficient expression of rCRP, and mutagenesis was necessary to correct the sequence.
  • some other features of the CRP coding sequence were identified which could hinder rCRP expression.
  • One of these is a potential stem-loop structure surrounding the site of the deletion.
  • a second is poor codon usage.
  • the mutagenesis therefore, had three goals: 1) to re-introduce the missing base into codon 47; 2) to reduce the possibility of mRNA secondary structure; and 3) to replace codons infrequently used in E. coli .
  • the mutagenic primer was designed with those goals in mind.
  • the target sequence for mutagenesis and the changes to be introduced are shown below.
  • the bold letter indicates the base that was inserted into the site of the deletion (codon 47) .
  • the underlined codons (48 and 50) are others that were changed for purposes of increasing expression.
  • oligonucleotide primers spanning the target sequence were synthesized by the phosphoramidite method on an Eppendorf Synostat-D synthesizer using Eppendorf reagents. The sequences of these primers are shown as A and B in Table 5 below. Two additional oligonucleotides (C and D) were synthesized for use as flanking primers. Their sequences and locations relative to the CRP sequence and a schematic diagram of each of the reactions performed are shown in Figure 7.
  • Reactions 1 and 2 illustrated in Figure 7 employed 10 nmol of Jfindlll-linearized pIT4 as template, 10 p ol of each primer, 0.2 mM deoxynucleotide triphosphates (Pharmacia) , 10 ⁇ l of lOx GeneAmp R reaction buffer (Perkin Elmer Cetus), and 2.5 units AmpliTaq R Taq DNA Polymerase (Perkin Elmer Cetus) in a total volume of 100 ⁇ l.
  • Reaction buffer contains 50 mM KCl, 10 mM Tris- HCl (pH 8.3), 1.5 mM MgCl 2 and 0.001% (w/v) gelatin (final concentrations) .
  • Reactions were carried out in a DNA Thermal Cycler Model 480 from Perkin Elmer Cetus. Following a three-minute incubation at 94°C, the unit was programmed to run 20 cycles of 94°C, 1 min.; 50°C, 1 min.; 74°C, 1 min., ending with a 7 min. incubation at 72°C.
  • PCR products were purified on a vertical 1.6% agarose gel (1.5 mm thick) run in TAE (Tris Acetate/EDTA) . Prior to casting the gel, plates, combs and spacers were soaked in 1 N HCl for 30 minutes to guard against contamination. The appropriate bands were excised and the PCR products purified using a GeneClean II R kit (BiolOl Inc.) according to the manufacturer's instructions. The DNAs were eluted in a volume of lOO ⁇ l TE (10 mM Tris-Cl, pH 8.0, 1 mM EDTA). The yields of DNA were estimated from the prep gel.
  • TAE Tris Acetate/EDTA
  • a second set of PCR reactions was performed to splice together the first-round products, thus restoring the full-length coding sequence.
  • Fifty ng of the 3' product and 20 ng of the 5' product were used as template, and 10 pmol each of primers A and B were used for amplification. Other conditions for the reactions were as in the first round.
  • Four thermal cycles were performed prior to the addition of the primers to give a chance for the extension reaction to begin. After primers were added, the reactions were continued for another 20 cycles.
  • the vector pETV and the final PCR product were digested with W el and BamHI.
  • the fragments were gel- purified using GeneClean.
  • the vector was then treated with calf intestinal alkaline phosphatase.
  • Vector and insert were ligated, and the ligation mixture was used to transform E. coli BL21(DE3).
  • Transformants were screened by minipreps performed as described in Sambrook et al., Molecular Cloning: A Laboratory Manual (2nd ed. , 1989, Cold Spring Harbor Laboratory Press) .
  • Analytical digests were performed on the miniprep DNA using Smal, Ndel and Kpnl , Ndel and BamHI , and XJal and Hindlll .
  • E. coli BL21(DE3) cells bearing pITIO were cultured to assess the effects of the modifications described in section F above on mutant rCRP expression.
  • Cells were grown at 37°C in a fourteen-liter New Brunswick Model FS614 fermentor in a medium containing NZ amine A R (enzymatic hydrolysate of casein; Quest International; 20 g/1) , (NH 4 ) 2 S0 4 (2 g/1) , KH 2 P0 4 (1.6 g/1), Na 2 HP0 4 :7H 2 0 (9.9 g/1), sodium citrate (0.65 g/1), MgS0 4 (0.24 g/1), glucose (22 g/1) and 100 ⁇ g/ml ampicillin.
  • the inclusion body protein was analyzed by SDS- PAGE and Western blot.
  • SDS-PAGE To perform the SDS-PAGE, the inclusion body protein was electrophoresed using the standard Pharmacia Phast electrophoresis system and reagents. In particular, commercially prepared SDS-PAGE buffer strips and 20% acrylamide gels (20% Phast gels) were used. Commercially available molecular weight standards were run in parallel wells to assess apparent molecular weights. Gels were stained for protein using Coomassie Brilliant Blue. Gels were subjected to Western blot analysis by transferring electrophoretically- separated protein onto nitrocellulose paper and adding monospecific goat anti-neo-CRP antiserum LP4-HRP (prepared as described in section F above) .
  • Plasmid stability was assessed by a simple plating test described in Studier and Moffat, J. Mol. Biol.. 189. 113-120 (1986). Briefly, prior to induction, samples are withdrawn and plated on LB medium, LB medium with ampicillin, LB medium with IPTG, and LB medium with ampicillin and IPTG. Since ampicillin resistance is conferred on E . coli BL21(DE3) by the ⁇ -lactamase gene on the plasmid, only cells bearing the plasmid will grow on LB with ampicillin. In contrast, cells with the plasmid grow poorly, if at all, on LB medium with IPTG, since rapid transcription of the target gene by T7 RNA polymerase occurs in the presence of IPTG.
  • Novagen, Inc. has developed several variants of the BL21(DE3) expression strain and of the pET3a vector used in the work described above. Eight additional expression strains utilizing some of these vectors (pET- 9a and pET-24a(+), as well as pET-3a) and E. coli strains (BLR(DE3) and BLR(DE3)pLysS, as well as BL21(DE3)) were constructed with the aims of increasing induced expression, decreasing basal transcription, and improving plasmid stability.
  • Plasmids pET-9a and pET-24a(+) are derivatives of plasmid pET-3a in which the ⁇ -lactamase gene has been replaced with the gene conferring resistance to kanamycin. Both plasmids possess the T7 promoter, but pET24a(+) has been further modified by inserting the lac operator sequence between the promoter and the multiple cloning site. This vector is designed to provide a binding site for the lac repressor which will, in the absence of inducer, prevent transcription of the target gene.
  • E. coli strain BLR(DE3) is identical to BL21(DE3), except that it is rec A " and is therefore recombination deficient.
  • Strain BLR(DE3)pLysS is a derivative of BLR(DE3) which carries a plasmid-borne copy of the T7 lysozyme gene. This strain is designed to provide a small quantity of T7 lysozyme to bind and inhibit any T7 RNA polymerase synthesized prior to induction.
  • both plasmids have the Xbal and BamRI sites in the same positions as in the pETV vector used in the previous expression systems.
  • the mutant CRP coding sequence was therefore excised from pITlO and cloned into each vector digested with the same enzymes.
  • the ligation mixtures were transformed into E. coli DH5 ⁇ cells, and transformants were selected by kanamycin resistance. Colonies were screened by restriction analysis of miniprep DNA (procedure described above in section F) . Plasmid DNAs from confirmed recombinants were purified on cesium chloride gradients. These plasmids were designated pIT12 (derived from pET-9a) and pIT13 (derived from pET-24a(+)). The preparation of pIT13 is shown in Figure 8.
  • Expression plasmids pITlO, pIT12 and pIT13 were independently transformed into E. coli BL21(DE3), BLR(DE3) or BLR(DE3)pLysS. Transformants were selected by resistance to appropriate antibiotics (see Table 6 below) , and the colonies screened by restriction analysis of miniprep DNA (procedure described above in section F) . Confirmed recombinants were expanded in liquid culture in LB medium with the following antibiotics: Table 6
  • Plasmid stability was determined as described above using the appropriate antibiotics (see Table 6) .
  • the greatest plasmid loss was observed in E . coli BL21(DE3) bearing pITlO which confers ampicillin resistance (-10%).
  • the extent of plasmid loss in E . coli BL21(DE3) was ⁇ 1%.
  • the rec A " strains BLR(DE3) and BLR(DE3)pLysS no plasmid loss was detected with either pIT12 or pIT13.
  • pITlO in E. coli BLR(DE3) about 3% of cells had lost plasmid, whereas this plasmid appeared stable in E. coli BLR(DE3)pLysS. Plasmid stability is set forth in Table 7 below.
  • a sample of a lysate of each culture was subjected to SDS-PAGE electrophoresis on a 20% Phast gel and subsequent Western blotting (procedure described above, this section) using mAb 3H12.
  • the lysates were prepared by suspending the cell pellets (obtained by centrifuging the 10 ml samples of each culture as described above) in 1 ml of 50 mM Tris-Cl, pH 8.0, 2 mM EDTA.
  • BLR(DE3)pLysS strains bear a plasmid encoding T7 lysozyme. In addition to its ability to bind to and inhibit the activity of T7 RNA polymerase, this protein digests the bacterial cell wall. Cells expressing this protein are somewhat more fragile than those that do not, and will lyse after freezing and thawing. While this has some advantages, our protocol for storing and resuspending the harvested cell paste on a large scale (see next section) would be complicated by this. Furthermore, the BLR(DE3)pLysS expression strains have two separate plasmids. In addition to the requirement for two antibiotics to maintain selection for both plasmids, the analysis of plasmids isolated from these strains is complicated by the fact that a mixture is always present.
  • the expression system chosen was plasmid pIT13 in E. coli BLR(DE3) .
  • the combination of the kanamycin resistance marker and the T7-lac operator promoter help to ensure that the plasmid will remain under selection and that very little, if any, expression will occur prior to induction.
  • the rec A ⁇ genotype of the strain limits the possibility of recombination.
  • the characteristics of the strain permit ease of handling on a production scale.
  • Plasmid pIT13 was isolated from the E. coli BLR(DE3) expression strain and purified on a cesium chloride gradient.
  • the CRP coding region was sequenced in the expression vector by the dideoxy chain terminator method of Sanger et al., Proc. Nat'l. Acad. Sci. USA. 74. 5463-5467 (1977) using Sequence version 2.0 (U.S. Biochemical Corp.) and 33 P-dATP as the radiolabel.
  • Figure 9 illustrates the sequencing strategy.
  • the plasmid is represented by the long narrow line in the center.
  • the start and stop codons and the restriction sites used for cloning into pET24a(+) are marked.
  • the primers are represented by small arrows, and the region of the sequence read from each primer is shown in bold.
  • Three forward (29f, 184f 446f) and three reverse (353r, 527r, 783r) primers were used to cover the entire coding region.
  • the dashed line is the region designed to be read by the 783r primer (see below) .
  • Reaction products were separated on a 5% Hydrolink Long Ranger gel (AT Biochem Corp.). A sample of each reaction mixture was loaded and electrophoresed until the bromphenol blue dye marker reached the bottom of the gel (1st load) . Identical samples were then applied to the remaining wells (2nd load) and electrophoresis continued until the bromphenol blue dye marker contained in the second set of samples reached the bottom of the gel. The gel was then dried and contacted with X-ray film for 2, 6.5, or 17 hours, and the sequence was read from the developed film.
  • E. coli BLR(DE3) bearing the pIT13 plasmid was cultured in a 250 liter pilot fermentor (New Brunswick Scientific) using the conditions described in the first paragraph of section G above, except that the culture medium contained 50 ⁇ g/ml kanamycin instead of the ampicillin. After the three-hour induction period, the cells were harvested by continuous flow centrifugation at 15,000 rpm in a Sharpies model AS16VB tubular bowl centrifuge. The harvested cell paste was transferred into sterile plastic bags and frozen at -80°C.
  • the cell paste was thawed by placing the sealed plastic bags in a 45°C water bath.
  • the thawed cell paste was then suspended in cold breakage buffer (20mM Tris- HCl, pH 7.6, 5mM EDTA, ImM phenylmethyl sulfonyl fluoride (PMSF; Sigma Chemical Co.) at a ratio of 200 ml buffer per gram cell paste and processed in a sterile blender at low speed for 30-45 seconds.
  • the homogeneous suspension was passed twice through a Nyro Soave homogenizer (equipped with a cell disruption valve) at a pressure of 500 bar. As it exited the homogenizer, the suspension was passed through a sterile cooling coil packed in an ice bath for cooling. It was then collected in a sterile covered container on ice.
  • This lysate was centrifuged at 12,000 x g in a Beckman model J2-21 centrifuge for 10 minutes at 4°C.
  • the pellet was resuspended in breakage buffer at 100 ml buffer per gram of initial cell paste.
  • the suspension was then passed twice through the homogenizer, this time at 700 bar.
  • the extract was centrifuged at 12,000 x g for 25 minutes at 4°C in a Beckman model J2-21 centrifuge to collect the inclusion body pellet.
  • the inclusion bodies were washed once with breakage buffer at a ratio of 150-200 ml buffer per 100 grams of initial cell paste by suspending the pellet in the buffer with a sterile glass rod and then centrifuging as described above. They were then washed three times with wash buffer (breakage buffer containing 0.5% Triton X-100 (Sigma Chemical Co.) at a ratio of 100-150 ml per 100 grams initial cell paste. The pellets were finally suspended in approximately 75 ml of breakage buffer per 100 grams of initial cell paste, aliquoted into sterile tubes, and frozen at -80°C until further processing.
  • FIGS. 10A and 10B Representative SDS-PAGE and Western blot analyses of three lots of inclusion bodies are shown in Figures 10A and 10B.
  • samples were diluted to approximately 1 mg/ml and boiled in SDS and ⁇ -mercaptoethanol, and 4 ⁇ l of each treated sample were loaded onto a homogenous 20% Phast gel.
  • the gel was either stained with Coomassie Brilliant Blue (Figure 10A) or the proteins were transferred to nitrocellulose and probed with monospecific goat anti-neo CRP antiserum LP4- HRP ( Figure 10B) .
  • the concentration of protein in the inclusion body suspension was estimated using the Bicinchoninic acid protein assay (Pierce Chemical Co.) or by solubilizing the protein in 6M GuHCl and measuring the A 280 (using the extinction coefficient for pure mCRP; 1.95[mg/ml] _1 ) .
  • the inclusion bodies were then pelleted by centrifuging for 10 minutes at 12,000 rpm in a Sorvall GSA rotor. The supernatant was discarded, and the pellet was dissolved in a solution of 6M GuHCl in 25 mM Tris, pH 8 (6M GuHCl/Tris) , to a concentration of between 5-10 mg protein/ml.
  • the concentration of solubilized protein was determined by measuring the A 280 of a diluted sample.
  • the inclusion body preparation was then diluted to a final concentration of 5 mg protein per ml with the 6M GuHCl/Tris buffer.
  • an initial ammonium sulfate fractionation step was performed. Using a peristaltic pump, a saturated solution of ammonium sulfate was added dropwise with stirring to a final concentration of 25% saturation at 0°C. The resulting suspension was stirred on ice for another 30 min following the completion of the ammonium sulfate addition, after which it was centrifuged 10 min at 12,000 rpm in a Sorvall GSA rotor. The supernatant, which contains primarily impurities, was discarded. The pellets were washed with sterile saline in a volume equivalent to that of the original solubilized protein solution to remove residual ammonium sulfate. Pellets were stored at 4°C until further processing.
  • a Q-Sepharose Fast Flow 11 anion exchange column was equilibrated with 25mM Tris-Cl, pH 8. The washed ammonium sulfate precipitate containing the mutant rCRP was solubilized at a concentration of 0.5 mg/ml by adding 10 mM Tris base and stirring until a suspension was formed. NaOH was then added until the solution became clear (pH 12.2-12.5). The pH was then titrated to 9.0 with HCl. The solubilized protein was loaded onto the Q- Sepharose Fast Flow R column at a linear flow rate of 30 cm/hour, with the total protein load not exceeding 5 mg/ml of resin.
  • the column was washed with 1 volume of the equilibration buffer (25mM Tris-Cl, pH 8), and then developed with a two-column volume linear gradient of 0-1 M NaCl in 25mM Tris-Cl, pH 8. After the NaCl concentration was rapidly returned to 0, the column was washed with an additional column volume of the equilibration buffer.
  • the mutant rCRP was finally eluted from the column with 8M GuHCl in 25mM Tris, pH 8.
  • a 280 was measured using the BioPilot R system.
  • a representative elution profile generated by the BioPilot R system is shown in Figure 11.
  • the mutant rCRP adsorbs very strongly to the Q- Sepharose Fast Flow 1 * column in the absence of a denaturant and will not elute with the NaCl gradient or with a pH gradient up to at least pH 12, allowing for an excellent separation of the mutant rCRP from endotoxin.
  • the mutant rCRP readily elutes from the column in the presence of a denaturant, such as GuHCl. However, urea should not be used for this elution step.
  • the eluate from the Q-Sepharose Fast Flow 1 * column was concentrated by precipitation with 25% saturated ammonium sulfate as described above. After the pellet was washed with saline, it was dissolved in 3% (w/v) sodium dodecyl sulfate (SDS) , 25 mM Tris-Cl, pH 9.0 at a concentration of 5-10 mg protein/ml (estimated using absorbance at 280 nm and extinction coefficient for pure mCRP) . It was then applied to a 5 cm x 92 cm Superdex 200 gel filtration column previously equilibrated in 1% SDS, 25 mM Tris-Cl, pH 8.0.
  • SDS sodium dodecyl sulfate
  • the sample size was 1-1.5% of the column volume, and the linear flow rate was 30 cm/hr.
  • the mutant rCRP typically eluted at a volume of 1120 ml (60% of the total bed volume) , and collection began when the peak reached 25- 30% of its expected height and was terminated when it returned to the same level.
  • a 280 was measured on the Biopilot R system.
  • Figure 12 illustrates a representative chromatogram for this column.
  • the mutant rCRP collected from the Superdex 200 column were first chilled to 0-4°C. Any precipitated SDS was then removed by a 5-10 min centrifugation at 12,000 rpm in a Sorvall GSA rotor. Then 5 ⁇ l of a 25% (w/v) solution of KC1 per ml of supernatant was added, and the insoluble potassium dodecyl sulfate was removed by centrifugation at 12,000 rpm in a Sorvall GSA rotor. The KC1 addition and centrifugation were repeated twice more.
  • the solubilized mutant rCRP was next applied to a Sephadex G-25 (fine) column (Pharmacia) equilibrated in 10 mM Tris-Cl, pH 7.4, at a linear flow rate of 70 cm/hr with the sample volume not to exceed 20% of the column volume.
  • a 280 and conductivity were measured on the Biopilot R system, and the mutant rCRP was usually present at a concentration of 1.5-2 mg/ml.
  • a typical chromatogram for this column is shown in Figure 13.
  • Figure 14 presents the SDS-PAGE results for the complete purification scheme and illustrates the increase in purity attained in each step.
  • the final mutant rCRP preparation is nearly homogenous.
  • samples were boiled in 2.5% SDS and 5% (v/v) ⁇ -mercaptoethanol, and 8 ⁇ g of each sample was loaded onto a 20% homogeneous Phast gel. Proteins were visualized by staining with Coomassie Brilliant Blue.
  • the concentration of residual SDS in the mutant rCRP preparation was measured using the acridine orange binding assay described in Anal. Biochem.. 118. 138-141 (1981) . Following the purification protocol described above, the concentration of residual SDS in the mutant rCRP preparation is routinely below the limit of detection (-0.001% w/v) of the assay.
  • the purified mutant rCRP was sterile filtered through 0.2 ⁇ filters.
  • the filtered, sterile 96/06624 PCMJS94/09729 mutant rCRP was then bottled in pyrogen-free sterile vials for injection.
  • Purified mutant rCRP produced by the method described in section H was compared with mCRP in SDS-PAGE and Western blot analyses using 20% Phast gels as described in section G above.
  • the mCRP was prepared from purified native CRP as described in section C above.
  • An ELISA assay was performed to determine if monoclonal antibodies specific for different epitopes on mCRP would react with the mutant rCRP produced by pIT13 and purified as described in section H.
  • the ELISA assay was performed as described in section D above using the following three monoclonal antibodies: 3H12, 8C10 and 7A8.
  • the preparation and properties of mAb 3H12 and 8C10 are described above in section D.
  • the preparation and properties of mAb 7A8 are described in U.S. Patent No. 5,272,257, PCT application WO 91/00872, Ying et al., J. Immunol.. 143. 221-228 (1989) and Ying et al. , Mol. Immunol..
  • Monoclonal antibody 3H12 reacts with the terminal octapeptide of mCRP, as noted above.
  • Monoclonal antibody 8C10 reacts with an epitope near the amino end of the mCRP sequence; this epitope is presumed to involve residues 22-45, which includes cysteine 36 which is mutated in the mutant rCRP. See Ying et al., Mol. Immunol.. 21, 677-687 (1992).
  • Monoclonal antibody 7A8 reacts with a third region of mCRP presumed to involve residues 130-138. See Ying et al., Mol. Immunol.. 2£, 677-687 (1992).
  • Biological Activity Modified-CRP has been characterized as a protein which selectively binds immune complexes and aggregated immunoglobulin.
  • the ability of the mutant rCRP produced by pIT13 and purified as described in section H above to bind aggregated immunoglobulin was evaluated and compared with the binding of aggregated immunoglobulin by mCRP. This evaluation was made using the ELISA assay described in section E above, except that only 0.2 ⁇ g of each test protein was immobilized per well.
  • Figure 17A shows that both mutant rCRP and mCRP bound aggregated IgG. Binding levels for the two proteins were approxima ⁇ tely equivalent.
  • PAP peroxidase-anti- peroxidase
  • mCRP and the full-length mutant rCRP subunits produced by pIT4 and pIT13 were digested with 2- (2'-nitrophenylsulfenyl)-3-methyl-3'-bromoindolenine (BNPS-Skatole; Pierce Biochemicals) which cleaves on the C-terminal side of tryptophan residues.
  • mCRP was prepared as described in section C above.
  • the full- length products of pIT4 and pIT13 were produced by culturing E. coli BL2l(DE3) and BLR(DE3) , respectively, and purifying the full-length products from the cultures, all as described above in sections G and H.
  • the mCRP and the final purified pIT4 and pIT13 products were solubilized at a concentration of 50 mg/ml in 25 mM Tris- Cl, pH 8, containing 6M GuHCl.
  • the reactions were initiated by the addition of four volumes of glacial acetic acid containing 25 mg/ml BNPS-Skatole.
  • the final reaction conditions were 10 mg/ml protein, 1.2 M GuHCl, 80% acetic acid and 20 mg/ml BNPS-Skatole.
  • 42.5 ⁇ l of each sample were transferred to an Eppendorf tube, and an equal volume of saturated ammonium sulfate was added to each tube.
  • Trp in the CRP sequence at position 67 which is also found in the Tenchini et al. sequence (see Table 3) . Cleavage at this site would produce a fragment of - 8000 Mr. As shown in Figure 18A, digestion of the mCRP and of the pIT13 product produced a prominent fragment of this size, whereas digestion of the pIT4 product produced a fragment of - 11,000 Mr, the expected size if Trp68 were absent. Since the pIT4 and pIT13 products were digested under identical conditions, these results strongly suggest that Trp68 is missing in the pIT4 product and that the out-of-frame sequence of pIT4 extends at least to codon 67.
  • the two mutant rCRP's were digested with endoproteinase LysC (Boehringer Mannheim Biochemicals) , which cleaves specifically at the C-terminal side of lysine residues.
  • the purified mutant rCRP's (prepared as described above, this section) were solubilized in 8 M urea, 25 mM Tris-Cl, pH 8.0, at a concentration of 5 mg/ml. Then, 0.1 M Tris-Cl, pH 7.4, enzyme and water were added in sufficient quantities to give final conditions of 25 mM Tris-Cl, pH 7.4, 4 M urea, 0.05 U enzyme, and 2.5 mg/ml protein.
  • the digests were incubated for 24 hours at room temperature, and the digestion products were analyzed by SDS-PAGE as described in section G above, except that high density gels designed for peptide separations were used.
  • SEQ ID NO:24 Residues shown in bold are those that likely differ from the bona fide CRP sequence and from the Tenchini et al. sequence (see Table 3 above) . Those residues which are underlined are still questionable in terms of their identity.
  • mutant rCRP's having amino acids in this region deleted or substituted or having amino acids added to this region can be prepared (by culturing host cells transformed with DNA coding for them) , and such mutant rCRP's should bind aggregated immunoglobulin or immune complexes as do the products of pIT4 and pIT13.

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Abstract

Cette invention concerne une protéine mutante comprenant la même séquence d'acides aminés qu'une sous-unité de protéine C-réactive (PCR) non mutée ou qu'une pré-PCR non mutée, sauf qu'au moins un acide aminé de la sous-unité PCR non mutée ou de la pré-PCR non mutée a été supprimé, qu'au moins un acide aminé de la sous-unité PCR non mutée ou de la pré-PCR non mutée a été remplacée par un autre acide aminé, qu'au moins un acide aminé a été ajouté à la sous-unité PCR non mutée ou à la pré-PCR non mutée, ou bien encore qu'on a effectuée une combinaison de ces changements. Le ou les acides aminés ajouté(s), supprimé(s) et/ou remplacé(s) sont sélectionnés de manière à ce que la protéine mutante soit moins susceptible de former des agrégats réticulés par covalence que la sous-unité PCR non mutée ou la pré-PCR non mutée. La protéine mutante présente également au moins une des activités biologiques de la CRP modifiée. Cette invention concerne également des molécules d'ADN qui codent pour les protéines mutantes de cette invention, des vecteurs qui expriment les protéines les protéines mutantes, des cellules hôtes qui ont été transformées afin de pouvoir exprimer les protéines mutantes et un procédé de production des protéines mutantes de cette invention qui consiste à mettre les cellules hôtes transformées en culture. Pour terminer, cette invention concerne des procédés et des matériaux permettant d'utiliser lesdites protéines mutantes.
PCT/US1994/009729 1994-08-26 1994-08-26 Proteine mutante, procedes et materiaux employes pour la produire et l'utiliser WO1996006624A1 (fr)

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US6251624B1 (en) 1999-03-12 2001-06-26 Akzo Nobel N.V. Apparatus and method for detecting, quantifying and characterizing microorganisms
US6429017B1 (en) 1999-02-04 2002-08-06 Biomerieux Method for predicting the presence of haemostatic dysfunction in a patient sample
US6502040B2 (en) 1997-12-31 2002-12-31 Biomerieux, Inc. Method for presenting thrombosis and hemostasis assay data
US6564153B2 (en) 1995-06-07 2003-05-13 Biomerieux Method and apparatus for predicting the presence of an abnormal level of one or more proteins in the clotting cascade
EP1409509A4 (fr) * 2001-07-25 2005-05-04 Isis Pharmaceuticals Inc Modulation antisens de l'expression de la proteine reactive c
US6898532B1 (en) 1995-06-07 2005-05-24 Biomerieux, Inc. Method and apparatus for predicting the presence of haemostatic dysfunction in a patient sample
US7179612B2 (en) 2000-06-09 2007-02-20 Biomerieux, Inc. Method for detecting a lipoprotein-acute phase protein complex and predicting an increased risk of system failure or mortality
US7211438B2 (en) 1999-02-04 2007-05-01 Biomerieux, Inc. Method and apparatus for predicting the presence of haemostatic dysfunction in a patient sample
US7425545B2 (en) 2001-07-25 2008-09-16 Isis Pharmaceuticals, Inc. Modulation of C-reactive protein expression
CN112218881A (zh) * 2018-11-29 2021-01-12 Cj第一制糖株式会社 cAMP受体蛋白变体及使用其制备L-氨基酸的方法

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Title
MOLECULAR IMMUNOLOGY, Volume 20, No. 11, issued 1983, POTEMPA et al., "Antigenic, Electrophoretic and Binding Alterations of Human C-Reactive Protein Modified Selectively in the Absence of Calcium", pages 1165-1175. *
THE JOURNAL OF BIOLOGICAL CHEMISTRY, Volume 260, No. 24, issued 25 October 1985, WOO et al., "Characterization of Genomic and Complementary DNA Sequence of Human C-Reactive Protein and Comparison With the Complementary DNA Sequence of Serum Amyloid P Component", pages 13384-13388. *
THE JOURNAL OF BIOLOGICAL CHEMISTRY, Volume 267, No. 35, issued 15 December 1992, AGRAWAL et al., "Probing the Phosphocholine-Binding Site of Human C-Reactive Protein by Site-Directed Mutagenesis", pages 25352-25358. *

Cited By (22)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6564153B2 (en) 1995-06-07 2003-05-13 Biomerieux Method and apparatus for predicting the presence of an abnormal level of one or more proteins in the clotting cascade
US6898532B1 (en) 1995-06-07 2005-05-24 Biomerieux, Inc. Method and apparatus for predicting the presence of haemostatic dysfunction in a patient sample
US6502040B2 (en) 1997-12-31 2002-12-31 Biomerieux, Inc. Method for presenting thrombosis and hemostasis assay data
US6429017B1 (en) 1999-02-04 2002-08-06 Biomerieux Method for predicting the presence of haemostatic dysfunction in a patient sample
US7211438B2 (en) 1999-02-04 2007-05-01 Biomerieux, Inc. Method and apparatus for predicting the presence of haemostatic dysfunction in a patient sample
US6251624B1 (en) 1999-03-12 2001-06-26 Akzo Nobel N.V. Apparatus and method for detecting, quantifying and characterizing microorganisms
US7179612B2 (en) 2000-06-09 2007-02-20 Biomerieux, Inc. Method for detecting a lipoprotein-acute phase protein complex and predicting an increased risk of system failure or mortality
US7425545B2 (en) 2001-07-25 2008-09-16 Isis Pharmaceuticals, Inc. Modulation of C-reactive protein expression
US8093224B2 (en) 2001-07-25 2012-01-10 Isis Pharmaceuticals, Inc. Antisense modulation of C-reactive protein expression
US7326693B2 (en) 2001-07-25 2008-02-05 Isis Pharmaceuticals, Inc. Antisense modulation of c-reactive protein expression
EP1409509A4 (fr) * 2001-07-25 2005-05-04 Isis Pharmaceuticals Inc Modulation antisens de l'expression de la proteine reactive c
US7491815B2 (en) 2001-07-25 2009-02-17 Isis Pharmaceuticals, Inc. Antisense modulation of C-reactive protein expression
US8710023B2 (en) 2001-07-25 2014-04-29 Isis Pharmaceuticals, Inc. Antisense modulation of C-reactive protein expression
US6964950B2 (en) 2001-07-25 2005-11-15 Isis Pharmaceuticals, Inc. Antisense modulation of C-reactive protein expression
US7915231B2 (en) 2001-07-25 2011-03-29 Isis Pharmaceuticals, Inc. Antisense modulation of C-reactive protein expression
EP2280019A1 (fr) * 2001-07-25 2011-02-02 ISIS Pharmaceuticals, Inc. Modulation antisens de l'expression de la protéine réactive C
US7863252B2 (en) 2003-06-02 2011-01-04 Isis Pharmaceuticals, Inc. Modulation of C-reactive protein expression
EP2266997A1 (fr) 2003-06-02 2010-12-29 Isis Pharmaceuticals, Inc. Modulation de l'expression de la protéine C-réactive
EP2218727A1 (fr) 2003-06-02 2010-08-18 Isis Pharmaceuticals, Inc. Modulation de l'expression de la protéine C-réactive
US8859514B2 (en) 2003-06-02 2014-10-14 Isis Pharmaceuticals, Inc. Modulation of C-reactive protein expression
CN112218881A (zh) * 2018-11-29 2021-01-12 Cj第一制糖株式会社 cAMP受体蛋白变体及使用其制备L-氨基酸的方法
CN112218881B (zh) * 2018-11-29 2023-11-07 Cj第一制糖株式会社 cAMP受体蛋白变体及使用其制备L-氨基酸的方法

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