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WO2000053777A1 - Elaboration de peptides par utilisation d'une sequence glucagon humain intervenant comme partenaire d'expression de fusion - Google Patents

Elaboration de peptides par utilisation d'une sequence glucagon humain intervenant comme partenaire d'expression de fusion Download PDF

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WO2000053777A1
WO2000053777A1 PCT/KR2000/000187 KR0000187W WO0053777A1 WO 2000053777 A1 WO2000053777 A1 WO 2000053777A1 KR 0000187 W KR0000187 W KR 0000187W WO 0053777 A1 WO0053777 A1 WO 0053777A1
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fusion
proteins
derivatives
kctc
preparing
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PCT/KR2000/000187
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Korean (ko)
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Young Hoon Park
Jeewon Lee
Dae-Young Kim
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Korea Research Institute Of Bioscience And Biotechnology
Hanwha Chemical Corporation
Ace Biotech
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Priority to AU34601/00A priority Critical patent/AU3460100A/en
Publication of WO2000053777A1 publication Critical patent/WO2000053777A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/52—Genes encoding for enzymes or proenzymes
    • 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
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/54—Interleukins [IL]
    • C07K14/55—IL-2
    • 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
    • C07K14/575—Hormones
    • C07K14/605—Glucagons
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion proteins
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/20—Fusion polypeptide containing a tag with affinity for a non-protein ligand
    • C07K2319/21—Fusion polypeptide containing a tag with affinity for a non-protein ligand containing a His-tag
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/70—Fusion polypeptide containing domain for protein-protein interaction
    • C07K2319/74—Fusion polypeptide containing domain for protein-protein interaction containing a fusion for binding to a cell surface receptor
    • C07K2319/75—Fusion polypeptide containing domain for protein-protein interaction containing a fusion for binding to a cell surface receptor containing a fusion for activation of a cell surface receptor, e.g. thrombopoeitin, NPY and other peptide hormones

Definitions

  • the present invention relates to a method for preparation of recombinant proteins or peptides expressed m form of X G -L-Y containing strongly self-associating peptide as fusion partner.
  • X G fusion partner (polypeptide containing whole or partial amino acid squence of human glucagon or its derivatives )
  • L linker (sequence for in-frame expression or m-vitro enzymatic cleavage)
  • Y recombinant protein or peptide
  • the present invention relates to genes encoding fusion proteins each of which comprises polypeptide containing whole or partial ammo acid sequence of human glu- cagon or its derivatives as a fusion partner, vectors comprising the genes, microorganisms transformed with the vectors, and methods for preparation of the fusion proteins.
  • a gene of target protein is inserted into an expression vector, and an appropriate host microorganism is transformed with this recombinant vector, and followed by culturing.
  • E . col i has been used as a host for production of useful foreign proteins by use of genetic recombinant technique because more informations about its genes and metabolic system are known than other microorganisms.
  • expression proteins In direct expression of target proteins m E. coli , expressed proteins must be transformed into natural proteins owing to addition of methionine to their N-terminus .
  • target proteins containing methionine at their N-terminus When target proteins containing methionine at their N-terminus are applied for human or other animals, lmmunogenecity can be induced, or structural instability of the proteins can cause their malfunction.
  • m expression of very toxic proteins, such as antibacterial peptide growth of host cells may be inhibited and stable large-scale production of small polypeptides less than 10,000 Da, is difficult owing to degradation by protenases in host cells.
  • the target proteins containing fusion prote ⁇ ns at N-terminus of target proteins could be produced with fusion partner which is expressed as soluble form in a selected.
  • these soluble proteins produced stably in E . col i are ⁇ -ga_--actos ⁇ dase , maltose binding protein (45 kDa), ⁇ 26 kDa), etc.
  • Fusion proteins using the aboie proteins as fusior partners can be purified easily chromatography, whereas recovery yield of polypeptides is decreased remarkably since the size of fusion partners are relatively larger than the desired polypeptides. Additionally, the above method cannot be used effectively in case that expressed fusion proteins maintain their cell toxicity.
  • target polypeptides are expressed in form of inclusion bodies in cells (Ulman, A. et al . , Gene, 29, 27-31, 1984; Nisson, B. et al . , EMBO J. 4, 1075-1080, 1985; Di Guan et al . , Gene 67, 2l- 30, 1988; La Vallie, E.R. et al . , Bio/Technology 11, 187-193, 1993) .
  • This method has an adventage in that the desired recombinant proteins can be isolated easily from cell culture in early state of isolation procedure.
  • the inclusion bodies is sometimes so non-specific that coaggregation of various host proteins (i.e.
  • Human glucagon is a peptide hormone that antagonizes msulm action and thereby leads to a stimulation of heptic glucose production.
  • the glucagon hormone is already commercialized as therapeutics for hypoglycemia or auxiliary for X-ray diagnosis and endoscopy America, Japan, etc. It is known that glucagon has 29 ammo acids and is stably produced in form of fusion protein at h gh expression rate in E . col i (Shin et al . , Appl . Microbiol . Biotechnol . 49, 364-370, 1998).
  • glucagon is small peptide (3.5 kDa)
  • X-ray analysis has demonstrated that in crystal, the peptide adopts a mainly ⁇ -helical conformation, and in dilute solution, the glucagon molecules have strong ten ⁇ ency to stabilize the ⁇ - helical conformation by hydrophobic interactions as an oligomer by self-association between molecules (Sasaki, K. et al . , Na t ure, 257, 751-757, 1979).
  • These properties of small-sized glucagon regarding mter olecular association provide a rationale for using glucagon as fusion expression partner for production of inclusion bodies containing the desired proteins at high purity as veil as for increment of production yield in cells.
  • the present inventors develope ⁇ a method for maximizing the production yield of recombinant prctsins cells, and purity in inclusion bodies by using polypec ide of human glucagon or its derivatives having strong tendency of self-association as an N-terminal fusion partner.
  • the method of the present invention enables inclusion bodies to solubilize easily in alkaline solution without using denaturants and reducing agents.
  • the method of the present invention makes it possible to recover and purify active proteins without using denaturants or reducing agents as well as to increase production yield in culture and purity of the desired proteins in inclusion bodies.
  • FIG. 1 represents a procedure for preparing expression vector, pT7IL-2,
  • FIG. 2 represents a procedure for preparing expression vector, pTlGIL-2,
  • FIG. 3 represents a procedure for preparing expression vector, pT2GIL-2,
  • FIG. 4 represents a procedure for preparing expression vector, pGHL-2
  • FIG. 5 represents a procedure for preparing expression vector, pG2IL-2
  • FIG. 6 represents a procedure for preparing expression vector
  • FIG. 7 represents intracellular expression level ⁇ %) of recombinant proteins produced from E. coli transformed with each vector
  • FIG. 8 represents content (purity, %) of recombinant proteins contained in insoluble inclusion bodies recovered from culture of each E. coli transformants
  • FIG. 9 represents solubility (%) of inclusion bodies recovered from culture of each E. coli transformants in alkaline solution
  • FIG. 10 represents results of western blotting after non-reducing SDS-PAGE of fusion proteins in inclusion bodies recovered from culture of each E. coli transformants
  • pTlGIL-2 lane 3 inclusion bodies produced from E . coli transformed with expression vector
  • pT2GIL-2 lane 4 inclusion bodies produced from E . coli transformed with expression vector
  • pGHL-2 lane 5 inclusion bodies produced from E .
  • FIG. 11 represents percentage of proteins formed by self-association between the recombinant protein molecules, as analyzed from results of western blotting after non- reducing SDS-PAGE of fusion proteins in inclusion bodies recovered from culture of each E. coli transformant .
  • the present invention provides a method for preparing the desired proteins of high purity at high yield from E . col i by using polypeptide containing amino acid sequence of human glucagon or its derivatives having strong tendency for self- association as a fusion partner.
  • the present invention provides a method for large-scale production of recombinant proteins in form of inclusion bodies by using heterologous polypeptides comprising hybrid peptide derived from human glucagon and tumor necrosis factor, or polypeptide containing 1 to 3-consecutive copies of human glucagon amino acid sequences as a fusion partner.
  • Partially modified glucagon derivatives as well as whole or partial ammo acids sequence of glucagon can be used for a fusion partner.
  • Whole or partial amino acids sequence of tumor necrosis factor or its mutant can be used as a fusion partner with glucagon, and polyhistidine sequence can be contained in the fusion partner to isolate easily recombinant proteins.
  • the present invention provides a method for preparing human proteins or its derivatives using microorganisms transformed with recombinant plasmids containing the above fusion partners sequence. Especially, the present invention provides a method for purifying recombinant proteins by renaturing the synthesized recombinant proteins through simply pH shift: the insoluble inclusion bodies of recombinant proteins produced by the above method in alkaline solution, followed by restoring pH to neutral condition.
  • the present invention produces recombinant proteins in form of fusion proteins using human glucagon peptide hormone (molecular weight 3.5 kDa) expressed stably in E . col i as a soluble protein.
  • Glucagon is a 29-residue peptide hormone having ⁇ - helical conformation, and expressed stably in E . col i . Moreover, glucagon has strong self-association tendency to stabilize the ⁇ -helical conformation by hydrophobic interaction forming an oligomer between molecules, ana thereby leads to a effective use as a fusion partner.
  • the present invention produces recombinant proteins by using polypeptides containing whole or partial human glucagon described by SEQ ID No: 1 as fusion partners.
  • the present invention provides a method for producing recombinant proteins using various peptides/polypeptides containing whole or partial ammo acid sequence of glucagon (G peptide) described by SEQ ID No: 1 as fusion partners.
  • N-termmus a polypeptide composed of peptide of 1 to 57 residues from N-termmus of human tumor necrosis factor (Tl peptide), G-peptide and polyhistidme [(H ⁇ s) e ] in order (a fusion partner TIG)
  • TIG N-termmus
  • T2G a polypeptide composed of peptide of 8 tr to 12 residues from N-termmus of human tumor necrosis factor
  • His polyhistidme
  • the present invention produces human interleukin-2 as a model protein in form of inclusion bodies at high expression level and high purity by using the above five fusion partners.
  • a method for construction of expression vectors producing the fusion proteins are a method for construction of expression vectors producing the fusion proteins.
  • the nucleotide sequence encoding human interleukin-2 is amplified by PCR (Polymerase Chain Reaction),
  • the amplified DNA is inserted into pT7-7 vector containing T7 promoter to construct expression vector pT7IL-2 capable of expressing the human interleukin-2, 3) Sequences encoding polypeptides, which are composed of the five fusion partners (TIG, T2G, Gl, G2, G3) having C- terminal Asp Asp Asp Asp Lys (DDDDK, hereinafter, referred as "D4K") , amino acid sequence recognized by enterokinase, are amplified by PCR, and 4) Expression vectors (pTlGIL-2, pT2GIL-2, pGIL-2, pG2IL, pG3IL-2) are constructed by insertion of the amplified DNA fragments into 5' -end of the interleukm-2 in pT7IL-2.
  • the expression vectors are capable of expressing fusion proteins composed of interleukin-2, D4K and the each five fusion partner, and the produced fusion proteins enables only interleukin-2 to be separated through digestion with enterokinse during purification procedure.
  • the DNAs encoding the polypeptides of the fusion partners having specific ammo acid sequence for enzymatic cleavage sequence by enterokinase at its C-termmus are inserted into vectors containing appropriate promoter, such as, lac, trp, tac, pL, T3, T7 , SP6, SV40, ⁇ (pL/pR) , etc., or the above DNAs (fusion partner-D4K) are ligated to DNAs having these promoters and ribosomal binding site, followed by construction of vector system through inserting them into various plasmids.
  • DNAs encoding the desired proteins or peptides are inserted into 3' -terminus of these vectors, the resulting expression vectors are introduced into appropriate hosts, and the constructed transformants are cultured to produce the desired fusion proteins.
  • the transformants were prepared by introducing the expression vectors producing the mterleukm-2 fusion proteins into suitable hosts by method of Hanahan (Hanahan, D. 1985, DNA cl oning 1, 109-135, IRS press) . Particulary, E . col i BL21(DE3) were transformed with the expression vector pTlGIL-2, pT2GIL-2, pGIL-2, pG2IL-2 and pG3IL-2, and the resulting transformants were deposited m Korean Collection for Type Cultures (KCTC) on November 13, 1998 (Accession No.: KCTC 0555BP, KCTC 0556BP, KCTC 0552BP, KCTC 0553BP, KCTC 0554BP) .
  • the fusion proteins containing mterleukm-2 can be produced b ⁇ cultu ⁇ ng the transformed recombinant E . col i under suitable conditions.
  • Cell extracts are obtained h) treatment with lysozyme digestion, freezing and thawing, ultrasonication or french press, followed by centrifugation or filtration.
  • the fusion proteins can be purified by general purification methods, such as solubilization of extracts, ultrafiltration, dialysis, ion exchange chromatography, gel filtration, electrophoresis and affinity chromatography.
  • Interleukm-2 is isolated by digestion with enterokinase .
  • the expression level of mterleukm-2 using fusion partners comprising human glucagon of the present invention is more excellently improved than the direct expression of mterleukm-2 (50-60% of total proteins in E . col i ) , and insoluble inclusion bodies containing foreign protein of high purity are obtained (about 80% of total proteins composing inclusion body) .
  • This insoluble inclusion bodies is solubilized more easily in alkaline solution than general insoluble inclusion bodies.
  • the general insoluble inclusion bodies needs solubilization using denaturants or reducing agent, followed by refolding procedure, whereas the fused recombinant proteins of the present invention are able to overcome the problems of refolding by simply solubilizmg the inclusion bodies in alkaline solution without using denaturants or reducing agents.
  • This property is due to that the N-termmal fusion partner causes binding of highly pure recombinant protein molecules by hydrophobic interaction between the same recombinant molecules EXAMPLES
  • Example 1 Construction of pT7lL-2 directly expressing i terleukin-2
  • E . coli by using T7 promoter expression system E . col i (KCTC 8258P) producing mterleukm-2 was obtained from Korean Collection for Type Culture.
  • Plasmid pNKM21 in this E . col i strain contains a structural gene for human mterleukm-2 mutant which has 125-cysteme substituted for se ⁇ ne, and this structural gene is regulated by PL promoter.
  • the sequence encoding 133 ammo acids of mterleukm-2 gene m pla id pNKM21 was amplified by PCR using the following primers . 1) GCA CAT ATG GCA CCT ACT TCA AGT TCT (pNIL)
  • PCR buffer (0.25 mM dNTPs; 50 mM KC1; 10 mM (NH 4 ) 2 S0 4 ; 20 mM T ⁇ s-HCl (pH 8.8); 2 mM MgS0 4 ; 0.1% Triton X- 100
  • PCR condition was 95°C/30 sec (denaturation) , 52°C/30 sec (annealing) , and 72°C/60 sec (elongation) (hereinafter, all PCRs were performed by this condition except specially mentioned PCR) . PCR was repeated by 30 cycles.
  • the amplified DNA fragments were analyzed on 1% agarose gel, purified by use of Quiagen Gel extraction kit, digested with Ndel and Hindlll, and the resulting D ⁇ A fragments having Ndel site at 5' -end and Hindi I I site at 3'- end were inserted into Nde I and Hind III sites of expression vector pT7-7.
  • the constructed recombinant plasmid was designated as pT7IL-2 (FIG. 1) .
  • Example 2 Construction of ⁇ nterleukm-2 expressing vector using human glucagon gene as a fusion partner
  • Tl peptide (l *f to 57 "" residue of human tumor necrosis factor) was amplified by PCR using mononuclear cell cD ⁇ A library (Clontech) constructed from
  • U937 cell line as a template, and the amplified D ⁇ A fragments were purified by Qiagen Gel extraction kit.
  • the purified D ⁇ fragments were digested with BamHI and Ndel to have Ndel site at 5' -end and BamHI sites at 3' -end, and inserted into ⁇ Jdel and BamHI siteS of a vector pT7-7. This resulting recombinant plasmid was designated as pT7-Tl.
  • Fragment-1 was amplified by PCR using the glucagon gene synthesized by D ⁇ A synthesizer as a template and the following primers.
  • Fragment-2 was amplified by PCR using the interleukin-2 gene in pT7IL-2 as a template and the following primers. 1) GAC GAC GAC GAC AAA GCA CCT ACT TCA AGT TCT (XID4K) 2) GCA AAG CTT CTA TTA AGT TAG TGT TGA GAT GAT (HIL)
  • the amplified D ⁇ A fragments were analyzed on 1% agarose gel, and purified by Quiagen Gel extraction kit.
  • Second PCR was performed by using fragment-1 and fragment-2 as templates and the following primers.
  • the amplified D ⁇ A fragments were analyzed on 1% agarose gel, and purified by gel extraction kit.
  • the purified D ⁇ A fragments were digested with -BamHI and Hindi 11 to have BamHI site at 5' -end and Hindi I I site at 3' -end, and inserted into BamHI and HindiII sites of pT7-Tl. This resulting recombinant plasmid was designated as pT7-TlGIL-2.
  • XH6IL-2 fragment was 'amplified by PCR using plasmid pT7-TlGIL as a template and the following primers.
  • the amplified DNA fragments were anlyzed on 1% agarose gel, and purified by gel extraction kit.
  • the purified DNA fragments were digested with Xhol and Hind ⁇ I I , respectively, to have Xho I site at 5' -end and HindiII site at 3' -end, and inserted into Xhol and HindiII sites of pT7-TlGIL-2.
  • This resulting recombinant plasmid was designated as pTlGIL-2 (FIG. 2) .
  • Ndel site and polyhistidine sequence [ (His) 10] /BamHI site were introduced at 5' and 3' ends of the coding sequence of Pro-Ser-Asp-Lys-Pro (8 th to 12 th residue of human tumor necrosis factor) , respectively, and the constructed D ⁇ A fragment was inserted into ⁇ Jdel and BamHI sites of pT7-7.
  • This resulting recombinant plasmid was designated as pT7-T2.
  • GIL-2 (BH) fragment was amplified by PCR using the gene of interleukin-2 fusion protein fused with glucagon in pT7-
  • T1GIL-2 as a template and the following primers.
  • HIL GCA AAG CTT CTA TTA AGT TAG TGT TGA GAT GAT
  • the amplified DNA fragments were analyzed on 1% agarose gel, and purified by gel extraction kit (Quiagen).
  • the purified DNA fragments were digested with BamH I and Hind III to have BamHI site at 5' end and Hindi I I site at 3' end, and inserted into BamHI and Hindi I I sites of pT7-T2. This resulting recombinant plasmid was designated as pT2GIL-2 (FIG.3) .
  • GH6IL-2 (BH) fragment was amplified by PCR using the gene of interleukin-2 fusion protein fused with glucagon in pGIL-2 as a template and the following primers .
  • G(NB) fragment was amplified by PCR using the gene of glucagon synthesized by DNA synthesizer as a template and the following primers.
  • the amplified DNA fragments were analyzed on 1.5% agarose gel, and purified by gel extraction kit (Quiagen) .
  • the purified DNA fragments were digested with Ndel and BamHI to have Ndel site at 5' end and BamHI site at 3' end, and inserted into Ndel and BamHI sites of pGIL-2 (BH) .
  • This resulting recombinant plasmid was designated as pG2IL-2(FIG. 5) .
  • G(EB) fragment was amplified by PCR using the gene of glucagon in pGIL-2 as a template and the following primers. 1) GCA GAA TCC CAC TCT CAG GGT ACT (GECO) 2) GCA GGA TCC AGT GTT CAT CAG CCA (GBAM-3)
  • the amplified D ⁇ A fragments were analyzed on 1.5% agarose gel, and purified by gel extraction kit.
  • the purified D ⁇ A fragments were digested with Eco- I and BamHI to have Hco-RI site at 5' end and BamHIsite at 3' end, and inserted into EcoRI and Ba HIsites of pGIL-2 (BH) .
  • This resulting recombinant plasmid was designated as pG2IL-2 (EH) .
  • G(NE) fragment was amplified by PCR using the gene of glucagon in pGIL-2 as a template and the following primers .
  • Example 4 Culture of E . coli transformants and production of fusion proteins
  • Example 5 Selection of E . coli having high productivity
  • the cells were distrupted by using Branson Somfier (Branson Ultrasonics Corp., Danbury, CT) . After separation of proteins in the lysates by SDS-PAGE (sodium dodecyl sulfate-polyacryamide gel electrophoresis) (14% Tris-Glycine gel), the expression level of each protein was • analyzed by using Densitometer (Bio-Rad) . From the result, colony showing the highest expression level was inoculated into LB and cultured. 15% glycerol was added to the culture in order to store at -70°C. The result of analysis of expression level demonstrated that expression of all fusion proteins was far superior in expression level to direct expression of only interleukin-2.
  • Example 6 Isolation of insoluble inclusion bodies from cell culture
  • the recombinant cells in culture were centrifuged at 6000 rpm and the cell pellets were resuspended in distilled water.
  • the cells were distrupted by using Branson Sonifier, centrifuged at 5000 g for 10 min, and the supernants and pellets were analyzed by SDS-PAGE (14% Tris-Glycine), respectively, in order to confirm recovery of proteins.
  • Insoluble protein aggregates were washed twice with 0.5% Triton X-100, and subject to necessary analyses.
  • Example 7 Construction of vectors expressing interleukin-2 using a human glucagon-derived gene as a fusion partner, and expression of fusion proteins by transformants
  • GK3IL-2 Recombinant interleukm-2 fused with glucagon mutant was constructed by the following method. Oligonucleotide GK3-1 and GK3-2 represented by SEQ ID No. : 2 and 3, respectively, were amplified by PCR using primers GNDE and GXHO.
  • the amplified DNA fragments were analyzed on 2% agarose gel, and purified by gel extraction kit (Quiagen).
  • the purified DNA fragments were digested with Ndel and Xhol to have Ndel site at 5' end and Xhol site at 3' end, and inserted into ⁇ del and Xhol sites of pIL-2 (NX) .
  • This resulting recombinant plasmid was designated pGK3IL-2.
  • pIL-2 was constructed by removing G3 sequence through digestion with Ndel and Xhol.
  • E . col i BL(DE3) was transforme ⁇ wit- pGK3lL-2, and the resulting E . col i transformant was deposite ⁇ in Korean Collection for Type Cell (KCTC) on February 24, 1999 (Accession NO : KCTC 0582BP) .
  • the colonies selected from the Example 5 were subcultured in 100 L of LB media containing 100 mg ampicillin per liter.
  • O.D. 600 reached 0.4
  • the gene expression was induced by adding IPTG (isopropylthio- ⁇ -D-galactosidase, 0.5 mM) , and the induced cultures were cultivated for further 3-4 hours under the same condition.
  • the insoluble protein inclusion bodies produced from the culture were recovered by using the method of the Example 6. After the recovered inclusion body was washed twice with 0.5% Triton X-100 and analyzed by SDS-PAGE (sodium dodecyl sulfate-polyacryamide gel electrophoresis) (14% Tris-Glycine gel). Purity of the recombinant protein in each inclusion body was estimated by- using densitometer (Bio-Rad) .
  • Insoluble proteins obtained from each recombinant strain were dried by using freeze-drymg method, resuspended n alkaline solution at pH 12 to the concentration of 50 mg/mL, and the solubility was estimated during sequential dilution (2X) using alkaline solution at pH 12) .
  • the sample was separated into a supernant and pellet through centrifugation (5000 g, 10 mm), and respectively quantitated by using Bradford method.
  • the solubiltity was determined by the following mathematical formula. The pellets were dissolved in solution at pH 12, and quantitated.
  • the ratio of hydrophobically-bound homologous multimer in each protein inclusion body was analyzed by using densitometer , and the result of analysis was shown in Figure 11.
  • the result showed that in direct expression of only mterleukm-2, hydrophobically-bound homologous multimer was 32%, however in expression in form of the fusion protein of the present invention, hydrophobically- bound homologous multimer was more than 60%, and plentifully above 90%.
  • Inclusion bodies in form of hydrophobic-bound homologous multimer were solubilized easily by using the above purification method.
  • polypeptides comprising ammo acid sequence of human glucagon provided by the present invention as fusion partners, recombinant proteins in inclusion bodies can be produced at high expression level, and simultaneously purity of recombinant proteins is able to be remarkbly improved.
  • the microorganism identified under I above was accompanied by:
  • microorganism identified under I above was received by this International Depositary Authority on and a request to convert the original deposit to a deposit under the Budapest Treaty was received by it on
  • the microorganism identified under I above was accompanied by:
  • the microorganism identified under I above was accompanied by:
  • microorganism identified under I above was received by this International Depositary Authority on and a request to convert the original deposit to a deposit under the Budapest Treaty was received by it on

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Abstract

La présente invention concerne l'élaboration de protéines ou de peptides de recombinaison exprimés sous forme de XG-L-Y contenant comme partenaire de fusion un puissant peptide d'auto-association. En l'occurrence, XG est partenaire de fusion, à savoir un polypeptide contenant une séquence d'acide aminé complète ou partielle de glucagon humain ou de ses dérivés. L est un lieur, à savoir une séquence apte à l'expression ou à la reconnaissance, dans le cadre, par une peptidase. Enfin, Y est protéine ou peptide de recombinaison. L'invention concerne plus particulièrement un gène codant pour une protéine de fusion qui comprend un polypeptide qui intervient comme partenaire de fusion et qui contient une séquence d'acide aminé complète ou partielle de glucagon humain ou de ses dérivés. L'invention concerne enfin un vecteur comprenant ce gène, un micro-organisme transformé par le vecteur, et un mode d'élaboration de la protéine de fusion.
PCT/KR2000/000187 1999-03-09 2000-03-09 Elaboration de peptides par utilisation d'une sequence glucagon humain intervenant comme partenaire d'expression de fusion WO2000053777A1 (fr)

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AU34601/00A AU3460100A (en) 1999-03-09 2000-03-09 Preparation of peptides by use of human glucagon sequence as a fusion expressionpartner

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KR1999/7641 1999-03-09
KR10-1999-0007641A KR100368073B1 (ko) 1999-03-09 1999-03-09 융합파트너를 이용한 재조합 단백질의 제조방법

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WO2002031103A1 (fr) * 2000-10-12 2002-04-18 Lee Seok Kun Dispositif et procede de regulation de l'expression genique a champ electromagnetique cyclique
EP1572908A2 (fr) * 2002-02-14 2005-09-14 William J. Rutter Molecules chimeriques permettant d'administrer un clivage a un hote traite
JP2009542203A (ja) * 2006-06-27 2009-12-03 コリア リサーチ インスティチュート オブ バイオサイエンス アンド バイオテクノロジー システインタグ付きブドウ球菌タンパク質ｇ変異体
WO2010064748A1 (fr) 2008-12-04 2010-06-10 Korea Research Institute Of Bioscience And Biotechnology Criblage de protéines abondamment sécrétées et leur emploi en tant que partenaires de fusion dans la production de protéines recombinantes

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KR20020003917A (ko) * 2000-06-26 2002-01-16 복성해 꼬리 단백질과 인간 인터루킨-16의 융합 단백질, 이 융합단백질을 코딩하는 유전자, 이 유전자를 포함하는 발현벡터, 이 벡터로 형질전환된 대장균 및 이 대장균을이용한 인간 인터루킨-16의 생산 방법
KR100980457B1 (ko) 2008-06-27 2010-09-07 한국생명공학연구원 대장균에서 활성형 인간 인터루킨-6의 생산 및 정제방법
JP2024545113A (ja) * 2022-05-23 2024-12-05 バクスディグム カンパニー，リミティド ウイルスヌクレオカプシドを用いた標的タンパク質発現プラットフォーム

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US5686268A (en) * 1992-06-19 1997-11-11 Pfizer Inc. Fused proteins
US6033877A (en) * 1995-11-02 2000-03-07 Research Foundation Of State University Of New York Peptide expression and delivery system

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US5506120A (en) * 1990-10-09 1996-04-09 M & D Research Co., Ltd. Method of producing peptides or proteins as fusion proteins
US5670340A (en) * 1991-08-19 1997-09-23 Suntory Limited Process for producing peptides in E. coli
US5686268A (en) * 1992-06-19 1997-11-11 Pfizer Inc. Fused proteins
US6033877A (en) * 1995-11-02 2000-03-07 Research Foundation Of State University Of New York Peptide expression and delivery system

Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002031103A1 (fr) * 2000-10-12 2002-04-18 Lee Seok Kun Dispositif et procede de regulation de l'expression genique a champ electromagnetique cyclique
EP1572908A2 (fr) * 2002-02-14 2005-09-14 William J. Rutter Molecules chimeriques permettant d'administrer un clivage a un hote traite
EP1572908A4 (fr) * 2002-02-14 2008-10-22 William J Rutter Molecules chimeriques permettant d'administrer un clivage a un hote traite
JP2009542203A (ja) * 2006-06-27 2009-12-03 コリア リサーチ インスティチュート オブ バイオサイエンス アンド バイオテクノロジー システインタグ付きブドウ球菌タンパク質ｇ変異体
US8541005B2 (en) 2006-06-27 2013-09-24 Korea Research Institute Of Bioscience And Biotechnology Cysteine-tagged streptococcal protein G variant
WO2010064748A1 (fr) 2008-12-04 2010-06-10 Korea Research Institute Of Bioscience And Biotechnology Criblage de protéines abondamment sécrétées et leur emploi en tant que partenaires de fusion dans la production de protéines recombinantes
EP2573122A2 (fr) 2008-12-04 2013-03-27 Korea Research Institute of Bioscience and Biotechnology Criblage de protéines abondamment sécrétées et leur emploi en tant que partenaires de fusion dans la production de protéines recombinantes
EP2918606A2 (fr) 2008-12-04 2015-09-16 Korea Research Institute of Bioscience and Biotechnology Criblage de protéines abondamment sécrétées et leur emploi en tant que partenaires de fusion dans la production de protéines recombinantes

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KR100368073B1 (ko) 2003-01-24
AU3460100A (en) 2000-09-28

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