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WO1997014786A1 - RECOMBINANT α-N-ACETYLGALACTOSAMINIDASE ENZYME - Google Patents

RECOMBINANT α-N-ACETYLGALACTOSAMINIDASE ENZYME Download PDF

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WO1997014786A1
WO1997014786A1 PCT/US1996/017466 US9617466W WO9714786A1 WO 1997014786 A1 WO1997014786 A1 WO 1997014786A1 US 9617466 W US9617466 W US 9617466W WO 9714786 A1 WO9714786 A1 WO 9714786A1
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ala
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PCT/US1996/017466
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French (fr)
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Alex Zhu
Jack Goldstein
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New York Blood Center, Inc.
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Priority to AU77196/96A priority Critical patent/AU7719696A/en
Publication of WO1997014786A1 publication Critical patent/WO1997014786A1/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
    • C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
    • C12Y302/01049—Alpha-N-acetylgalactosaminidase (3.2.1.49)
    • 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
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)

Definitions

  • This invention relates to a recombinant enzyme for use in the removal of type A antigens from the surface of cells in blood products, thereby converting certain sub-type A blood products to type 0 blood products and certain type AB blood products to type B blood products.
  • This invention further relates to methods of cloning and expressing said recombinant enzyme. More particularly, this invention is directed to a recombinant chicken liver ⁇ -N-acetylgalacto- saminidase enzyme, methods of cloning and expressing said
  • the recombinant ⁇ -N-acetylgalactosaminidase enzyme of this invention provides a readily available and cost-efficient enzyme which can be used in the removal of type A antigens from the surface of cells in type A and AB blood products.
  • Treatment of certain sub-type A blood products with the recombinant enzyme of this invention provides a source of cells free of the A antigen, which blood products are thereby rendered useful in transfusion therapy in the same manner of O type blood products.
  • blood products includes whole blood and cellular components derived from blood, including erythrocytes (red blood cells) and platelets.
  • blood group or type systems, one of the most important of which is the ABO system.
  • This system is based on the presence or absence of antigens A and/or B. These antigens are found on the surface of erythrocytes and on the surface of all endothelial and most epithelial cells as well.
  • the major blood product used for transfusion is erythrocytes, which are red blood cells containing hemoglobin, the principal function of which is the transport of oxygen.
  • Blood of group A contains antigen A on its erythrocytes.
  • blood of group B contains antigen B on its erythrocytes.
  • Blood of group AB contains both antigens
  • blood of group O contains neither antigen.
  • the blood group structures are glycoproteins or glycolipids and considerable work has been done to identify the specific structures making up the A and B determinants or antigens. It has been found that the blood group specificity is determined by the nature and linkage of monosaccharides at the ends of the carbohydrate chains.
  • the carbohydrate chains are attached to a peptide or lipid backbone which is embedded in the lipid bi-layer of the membrane of the cells.
  • the most important (immuno-dominant or immuno-determinant) sugar has been found to be
  • N-acetylgalactosamine for the type A antigen and galactose for the type B antigen There are three recognized major sub-types of blood type A. These sub-types are known as A 1; A intermediate (A int ) and A 2 . There are both quantitative and qualitative differences which distinguish these three sub-types. Quantitatively, k ⁇ erythrocytes have more antigenic A sites, i.e., terminal N-acetylgalactosamine residues, than A int erythrocytes which in turn have more antigenic A sites than A 2 erythrocytes.
  • the transferase enzymes responsible for the formation of A antigens differ biochemically from each other in A ⁇ A int and A 2 individuals. Some A antigens found in A x cells contain dual A antigenic sites. Blood of group A contains antibodies to antigen B.
  • blood of group B contains antibodies to antigen A.
  • Blood of group AB has neither antibody, and blood group 0 has both.
  • a person whose blood contains either (or both) of the anti-A or anti-B antibodies cannot receive a transfusion of blood containing the corresponding incompatible antigen(s) . If a person receives a transfusion of blood of an incompatible group, the blood transfusion recipient's antibodies coat the red blood cells of the transfused incompatible group and cause the transfused red blood cells to agglutinate, or stick together. Transfusion reactions and/or hemolysis (the destruction of red blood cells) may result therefrom.
  • transfusion blood type is cross-matched against the blood type of the transfusion recipient.
  • a blood type A recipient can be safely transfused with type A blood which contains compatible antigens.
  • type 0 blood contains no A or B antigens, it can be transfused into any recipient with any blood type, i.e., recipients with blood types A, B, AB or 0.
  • type 0 blood is considered "universal", and may be used for all transfusions.
  • the process for converting A int and A 2 erythrocytes to erythrocytes of the H antigen type which is described in the '627 Patent includes the steps of equilibrating certain sub-type A or AB erythrocytes, contacting the equilibrated erythrocytes with purified chicken liver ⁇ -N-acetylgalacto ⁇ saminidase enzyme for a period sufficient to convert the A antigen to the H antigen, removing the enzyme from the erythrocytes and re-equilibrating the erythrocytes.
  • ⁇ -N-acetylgalactosaminidase obtained from an avian liver (specifically, chicken liver) source was found to have superior activity in respect of enzymatic conversion or cleavage of A antigenic sites.
  • a recombinant, cloned enzyme allows for specific protein sequence modifications, which can be introduced to generate an enzyme with optimized specific activity, substrate specificity and pH range.
  • ⁇ -N-acetylgalactosaminidase enzymes are characterized (and thereby named) by their ability to cleave N-acetylgalactosamine sugar groups. In isolating or identifying these enzymes, their activity is assessed in the laboratory by evaluating cleavage of synthetic substrates which mimic the sugar groups cleaved by the enzymes, with p-nitrophenylglycopyranoside derivatives of the target sugar groups being commonly used.
  • these synthetic substrates are simple structurally and small-sized and mimic only a portion of the natural glycoproteins and glycolipid structures which are of primary concern, those being the A antigens on the surface of cells.
  • a natural glycolipid substrate originally isolated from sheep erythrocytes, is the Forsmann antigen (globopentaglycosylceramide) .
  • the Forsmann antigen substrate appropriately mimics the natural A antigen glycolipid structures and is therefore utilized to predict the activity of ⁇ -N-acetylgalactosaminidase enzymes against the A antigen substrate.
  • Isolated Forsmann antigen glycolipids have been shown to inhibit hemolysis of sheep red cells by immune rabbit anti-A serum in the presence of serum complement.
  • ⁇ -N-acetylgalactosaminindase enzyme has been isolated from a number of sources besides chicken liver (described above) , including bacteria, mollusks, earthworms, and human liver.
  • the human ⁇ -N-acetylgalactosaminidase enzyme has been purified, sequenced, cloned and expressed.
  • Human ⁇ -N-Acetylgalactosaminidase Molecular Cloning, Nucleotide Sequence and Expression of a Full-length cDNA by Wang et al., in The Journal of Biological Chemistry. Vol. 265, No. 35, pages 21859-21866 (December 15, 1990)
  • the cDNA encoding human ⁇ -N-acetyl ⁇ galactosaminidase was sequenced.
  • WO 92/07936 discloses the cloning and expression of the cDNA which encodes human ⁇ -N-acetylgalactosaminidase. Although human ⁇ -N-acetylgalactosaminidase has been purified, sequenced, cloned and expressed, it is not appropriate for use in removing A antigens from the surface of cells in blood products. In determining whether an enzyme is appropriate for use in removing A antigens from the surface of cells, one must consider the following enzyme characteristics, particularly with respect to the Forsmann antigen substrate: substrate specificity, specific activity or velocity of the substrate cleavage reaction, and pH optimum.
  • Substrate specificity is measured in the Km value, which measures the binding constant or affinity of an enzyme for a particular substrate.
  • the lower a Km value the more tightly an enzyme binds its substrate.
  • the velocity of an enzyme cleavage reaction is measured in the Vmax, the reaction rate at a saturating concentration of substrate. A higher Vmax indicates a faster cleavage rate.
  • the ratio of these two parameters, Vmax/Km is a measure of the overall efficiency of an enzyme in reacting with (cleaving) a given substrate. A higher Vmax/Km indicates greater enzyme efficiency.
  • the enzyme For successful and clinically applicable removal of A antigens from the surface of cells, the enzyme must be sufficiently active at or above a pH at which the cells being treated can be maintained.
  • Vmax/Km value for the Forsmann antigen of human a-N-acetylgalactosaminidase is 0.46, as compared to a Vmax/Km value of 5.0 for the chicken liver enzyme, indicating an approximately ten-fold difference in efficiency.
  • the Km is lower and the Vmax is higher for the chicken liver enzyme, compared to the human enzyme.
  • human ⁇ -N-acetylgalactosaminidase has a pH optimum for the Forsmann antigen of 3.9, compared to 4.7 for chicken liver ⁇ -N-acetylgalactosaminidase.
  • human ⁇ -N-acetylgalactosaminidase enzyme is not suitable for removal of A antigens, particularly when compared to the chicken liver enzyme.
  • Figure 1 represents a diagram of the strategy used to clone and sequence the chicken liver ⁇ -N-acetylgalacto ⁇ saminidase cDNA
  • Figure 2 represents the nucleic acid sequence and the deduced amino acid sequence of the chicken liver ⁇ -N-acetylgalactosaminidase cDNA clone;
  • Figure 3 represents the expression of chicken liver ⁇ -N-acetylgalactosaminidase in bacteria and rabbit reticulocyte lysate a ⁇ shown by Western blot;
  • Figure 4 represents a homology comparison between ⁇ -N-acetylgalactosaminidases and a-galactosidases
  • Figure 5 represents the expression of chicken liver ⁇ -N-acetylgalactosaminidase in yeast as shown by Western blot.
  • Figures 6A and 6B represent the determination of the molecular mass of the recombinant ⁇ -N-acetylgalacto ⁇ saminidase enzyme produced by the Pichia pastoris expression system in comparison to the native ⁇ -N-acetylgalacto- saminidase enzyme.
  • Figure 7 represents the results of the N- glycosidase treatment of the recombinant ⁇ -N-acetyl ⁇ galactosaminidase enzyme produced by the Pichia pastoris expression system and the native ⁇ -N-acetylgalactosaminidase enzyme.
  • Lanes 1 and 3 correspond to the untreated recombinant and native enzymes, respectively
  • lanes 2 and 4 correspond to the N-glycosidase F treated recombinant and native enzymes, respectively.
  • the labels a, b and c on the right side of the blot correspond to the recombinant enzyme, the native enzyme and both deglycosylated enzymes, respectively.
  • This invention is directed to a recombinant chicken liver ⁇ -N-acetylgalactosaminidase enzyme, which enzyme has a molecular weight of about 45 kDa, is immunoreactive with an antibody specific for chicken liver ⁇ -N-acetylgalactosaminidase, and also has about 80% amino acid sequence homology with human ⁇ -N-acetylgalacto ⁇ saminidase enzyme.
  • the recombinant chicken liver ⁇ -N-acetylgalactosaminidase enzyme of this invention has the amino acid sequence depicted in Figure 2, from amino acid number 1 to amino acid number 406.
  • This invention is further directed to methods of cloning and expressing the recombinant chicken liver ⁇ -N-acetylgalactosaminidase enzyme, and to a method of using said enzyme to remove A antigens from the surface of cells in blood products so as to convert said blood products of certain A sub-types to type O, thereby rendering said blood products universal for use in transfusion therapy.
  • This invention is directed to a recombinant enzyme for use in the removal of type A antigens from the surface of cells in blood products, thereby converting certain sub-type A blood products to type 0 blood products and certain sub-type AB blood products to type B blood products.
  • the recombinant chicken liver ⁇ -N-acetylgalactosaminidase enzyme of this invention has a molecular weight of about 45 kDa and is immunoreactive with an antibody specific for chicken liver ⁇ -N-acetylgalactosaminidase.
  • the recombinant enzyme of this invention has about 80% amino acid sequence homology with human ⁇ -N-acetylgalacto ⁇ saminidase enzyme.
  • a DNA vector containing a sequence encoding chicken liver ⁇ -N-acetylgalactosaminidase was deposited under the Budapest Treaty with the American Type Culture Collection, Rockville, Maryland, on March 17, 1993, tested and found viable on March 22, 1993 and catalogued as ATCC No. 75434.
  • the recombinant chicken liver ⁇ -N- acetylgalactosaminidase enzyme of this invention can be cloned and expressed so that it is readily available for use in the removal of A antigens from the surface of cells in blood products.
  • the enzyme of this invention can be cloned and expressed by screening a chicken liver cDNA library to obtain the cDNA sequence which encodes the chicken liver ⁇ -N-acetylgalactosaminidase, sequencing the encoding cDNA once it is determined, cloning the encoding cDNA and expressing ⁇ -N-acetylgalactosaminidase from the cloned encoding cDNA.
  • This may be performed by obtaining an amplified human ⁇ -N-acetylgalactosaminidase fragment capable of use as a screening probe, screening a chicken liver cDNA library, such as the one described hereinabove, using the amplified human ⁇ -N-acetylgalactosaminidase fragment as a probe so as to obtain the cDNA sequence of the chicken liver cDNA library which encodes chicken liver ⁇ -N-acetylgalacto ⁇ saminidase, sequencing the encoding DNA, cloning the encoding DNA and expressing chicken liver ⁇ -N-acetylgalacto ⁇ saminidase enzyme from the cloned encoding cDNA.
  • screening can be performed using antibodies which recognize chicken liver ⁇ -N-acetylgalactosaminidase.
  • Methods which are well known to those skilled in the art can be used to construct expression vectors containing the chicken liver ⁇ -N-acetylgalactosaminidase coding sequence, with appropriate transcriptional/ translational signals for expression of the enzyme in the corresponding expression systems.
  • Appropriate organisms, cell types and expression systems include: cell-free systems such as a rabbit reticulocyte lysate system, prokaryotic bacteria, such as E.
  • coli eukaryotic cells, such as yeast, insect cells, mammalian cells (including human hepatocytes or Chinese hamster ovary (CHO) cells) , plant cells or systems, and animal systems including oocytes and transgenic animals.
  • mammalian cells including human hepatocytes or Chinese hamster ovary (CHO) cells
  • plant cells or systems including oocytes and transgenic animals.
  • animal systems including oocytes and transgenic animals.
  • the entire chicken liver ⁇ -N-acetylgalacto ⁇ saminidase coding sequence or functional fragments of functional equivalents thereof may be used to construct the above expression vectors for production of functionally active enzyme in the corresponding expression system. Due to the degeneracy of the DNA code, it is anticipated that other DNA sequences which encode substantially the same amino acid sequence may be used.
  • changes to the DNA coding sequence which alter the amino acid sequence of the chicken liver ⁇ -N-acetylgalactosaminidase enzyme may be introduced which result in the expression of functionally active enzyme.
  • amino acid substitutions may be introduced which are based on similarity to the replaced amino acids, particularly with regard to the charge, polarity, hydrophobicity, hydrophilicity, and size of the side chains of the amino acids.
  • Sub-type A antigens can be removed from the surface of erythrocytes by contacting the erythrocytes with the recombinant chicken liver ⁇ -N-acetylgalactosaminidase enzyme of this invention for a period of time sufficient to remove the A antigens from the surface of the erythrocytes.
  • Chicken liver ⁇ -N-acetylgalactosaminidase was purified to homogeneity.
  • the enzyme was a glycoprotein with a molecular weight of 80 kDa, and was dissociated into two identical subunits at pH 7.5. Its optimal pH for cleavage of the synthetic p-nitrophenyl- ⁇ -N-acetylgalactosaminyl- pyranoside substrate was 3.65 and the activity dropped sharply when the pH was raised above 7.
  • N-terminal sequence obtained from the purified chicken liver a-N-acetylgalactosaminidase showed a strong homology with the corresponding sequence deduced from the human a-N-acetylgalactosaminidase cDNA clone described in Tsuji et al., and Wang et al.
  • a DNA fragment corresponding to human ⁇ -N-acetylgalactosaminidase residues from 688 to 1236 was amplified from the cDNA by the hot-start PCR technique.
  • the PCR reaction mixture was preheated at 95°C for 5 minutes and maintained at 80°C while Taq DNA polymerase (Promega) was added to reduce the possible non-specific annealing at lower temperature. 35 cycles of amplification was then carried out as follows: 94°C for 1 minute, 50°C for 2 minutes and 72°C for 3 minutes. The same conditions for PCR were applied in all of the following experiments.
  • the PCR-amplified fragment was then used as a radioactively-labeled probe in the screening of a chicken liver cDNA library (Stratagene) based on homology hybridization.
  • the filters containing the library were hybridized with the probe overnight at 42°C in a solution of 50% formamide, 5XSSPE, 5XDenhardt's, 0.1% SDS and 0.1 mg/ml salmon sperm DNA. The filters were then washed as follows:
  • FIG. 1 represents a diagram of the strategy used to clone and sequence the chicken liver ⁇ -N-acetylgalactosaminidase cDNA.
  • the cDNA encoding chicken liver ⁇ -N-acetylgalactosaminidase contained a 1.2 kb coding region (slashed area) and a 1.2 kb 3' untranslated region.
  • the arrows at the bottom of the diagram indicate the sequencing strategy.
  • CL1, CL2 and CL3 are oligonucleotides used as primers for the nested PCR.
  • CL1 and CL2 are located at position 924-941 nt and 736-753 nt, respectively (see Figure 2) .
  • the oligonucleotide CL3 [5'-CTGGAGAAC(T)GGA(GC)CTGGCT(CA)CG] was designed taking into account chicken codon usage and "best guess".
  • CL1 specific primer
  • CL2 universal primer derived from the library vector
  • the primer CL2 had the sequence located upstream of CL1 ( Figure 1) and the second primer, CL3, was designed based on the N-terminal amino acid sequence from purified chicken liver ⁇ -N-acetylgalacto ⁇ saminidase (see Figure 1) .
  • a 750 bp fragment was sequenced to eliminate any possible PCR artifacts. Since the 750 bp fragment overlapped with the 1.9 kb clone isolated by the library-screening, the two fragments were linked together by PCR to reconstitute the cDNA encoding chicken liver ⁇ -N-acetylgalactosaminidase ( Figure 1) .
  • the DNA sequencing was performed according to standard procedure, and the coding region was sequenced in both orientations.
  • Figure 2 represents the nucleic acid sequence and deduced amino acid sequence of the chicken liver ⁇ -N-acetylgalactosaminidase cDNA clone.
  • the underlined regions in Figure 2 match sequences obtained from the N-terminus and CNBr-derived fragments of enzyme purified from chicken liver.
  • the first 3 nucleotides, ATG, were added during subcloning to serve as the translational initiation codon for protein expression.
  • the polyadenylation signal (AATAAA) at positions 2299-2304 nt is double-underlined.
  • the boxed sequence indicates potential sites for N-glycosylation.
  • the mature protein of 405 amino acids has a molecular mass of about 45 kDa, consistent with that of the purified enzyme estimated by SDS-PAGE. Due to the cloning approach applied, the sequence at the 5' end of the cDNA corresponded to the N-terminal sequence of the mature enzyme isolated from chicken liver.
  • the sequence from 1 to 1260 nucleotides which contained the coding region for chicken liver a-N-acetylgalactosaminidase was subcloned into the vector PCR-II (Invitrogen) in such an orientation that the T7 promoter was located upstream of the insert. Since the N-terminus of the mature protein started with leucine, a translational initiation codon, ATG, was added during the subcloning construction. The construct was then used as a template in a transcription-translation coupled system, TNT system (Promega) , for protein expression according to the procedure recommended by the manufacturer.
  • TNT system Promega
  • the cDNA was subcloned into the EcoRI site of the pTrcHis vector (Invitrogen) for expression in E. coli. Because of the sequence in the vector, the expressed enzyme contained a polyhistidine-tag in its N-terminus, which permitted one step purification by affinity chromatography from crude cell lysates.
  • Figure 3 represents the expression of chicken liver ⁇ -N-acetylgalactosaminidase in bacteria and rabbit reticulocyte lysate as shown by Western blotting.
  • Lane 1 through lane 4 demonstrate the results of expression in a rabbit reticulocyte lysate.
  • the expression was carried out in lysate in the presence of 35 S-methionine with (lane 1) or without (lane 2) the expression plasmid.
  • 5 ml of the reaction sample was loaded to a 12% SDS-PAGE.
  • the gel was dried and autoradiographed for 2 hours and a band of an apparent molecular weight of about 45KDa was visualized with the expression plasmid (lane 1, Figure 3) .
  • a Western blot was performed using a polyclonal antibody raised against ⁇ -N-acetylgalactosaminidase purified from chicken liver.
  • the chicken liver ⁇ -N-acetylgalactosaminidase * sequence was compared with published sequences of other ⁇ -N-acetylgalactosaminidases and ⁇ -galactosidases which cleave ⁇ -galactose sugar groups.
  • Figure 4 shows a homology comparison between various ⁇ -N-acetylgalactosaminidases and ⁇ -galactosidases. Alignment was carried out using both the computer program PROSIS (Hitachi Software Engineering Corp., Ltd.) and manual arrangement. The amino acid sequences were deduced from cDNAs.
  • Sequences I and II are of ⁇ -N-acetylgalactosaminidases from chicken liver and human placenta, respectively.
  • Sequences III, IV, V and VI represent ⁇ -galactosidase from human, yeast, Cvamopsis tetragonoloba and Aspergillus niger. respectively.
  • Sequences IV and VI are truncated at the C-terminus, as indicated by **. Identical or conservatively substituted amino acid residues (five out of six or more) among the aligned protein sequences are boxed. The numbers above the sequences indicate the relative position of each peptide sequence.
  • the deduced amino acid sequence from chicken liver ⁇ -N-acetylgalactosaminidase cDNA shows approximately 80% homology with the human ⁇ -N-acetylgalactosaminidase as determined by PROSIS. This homology indicates the relatedness of the human and chicken liver enzymes, despite the differences in the specific characteristics of the enzymes, particularly with regard to cleavage of the Forsmann antigen, as has already been described. Also, polyclonal antibodies raised against chicken liver ⁇ -N-acetylgalactosaminidase enzyme do not cross react with the human enzyme. The specific amino acids responsible for these differences remain to be elucidated.
  • Yamachi et al. (1990) reported that a human ⁇ -N-acetylgalactosaminidase cDNA with an insertion of 70bp at the position corresponding to number 376 in Figure 4 was not enzymatically active in a transient expression study in COS cells.
  • the data suggests that the open reading frame shift caused by this insertion in the C-terminal portion of the molecule is responsible for the loss of enzymatic activity, indicating that amino acids in the C-terminal region may be essential for ⁇ -N-acetylgalactosaminidase enzyme activity.
  • the first 48 nucleotides of human ⁇ -N-acetyl ⁇ galactosaminidase cDNA (Wang, et al. 1990) which correspond to the signal peptide sequence, were linked to the cloned chicken liver ⁇ -N-acetylgalactosaminidase coding region by PCR.
  • the PCR amplified product was subcloned directly into the vector PCR-II (Invitrogen) .
  • Two EcoRI sites flanking the insert were used to subclone the entire ⁇ -N-acetyl ⁇ galactosaminidase cDNA into the yeast expression vector pYES2 (Invitrogen) in such an orientation that the GAL 1 promoter was located upstream of the insert.
  • the GAL 1 promoter provides expression of the inserted cDNA clone under galactose inducing growth conditions in yeast.
  • the yeast vector constructs were transformed into the yeast strain, INVSCI (Invitrogen) using standard procedures.
  • INVSCI Invitrogen
  • the total proteins from cell extract and culture supernatant were prepared and separated by 12% SDS-PAGE and a Western blot performed (by standard conditions) using the polyclonal antibody raised against purified chicken liver ⁇ -N-acetylgalactosaminidase.
  • the transformed yeast cells were grown in medium without uracil (Bio 101, Inc.). After 0.2% galactose induction, the cells were centrifuged and protein extracts were prepared using glass bead disruption. The secreted proteins in the culture supernatant were concentrated with a Centricon-30
  • Lanes 1 and 8 of Figure 5 show the ⁇ -N-acetylgalactosaminidase purified from chicken liver.
  • Lane 2 through lane 4 are cell extracts from the yeast transformed with three different pYES2 constructs: the vector alone (lane 2) , chicken liver ⁇ -N-acetylgalacto ⁇ saminidase cDNA coding region (lane 3) , and the coding region plus signal sequence (lane 4) .
  • Lane 5 is the culture supernatant from transformed yeast used in Lane 4.
  • Lane 7 shows the molecular weight standard. As shown in Figure 5, while the protein without signal peptide was expressed within yeast cells (lane 3) , the protein with a signal peptide sequence was predominantly secreted into the media (lane 5) .
  • the expressed enzyme eluted from the column demonstrates activity toward the synthetic substrate p-nitrophenyl- ⁇ -N-acetylgalactosaminylpyranoside at pH 3.6. Heavily glycosylated enzyme did not bind to the affinity column and showed no activity against synthetic substrate. All the data taken together demonstrate production, secretion and purification of enzymatically active chicken liver ⁇ -N-acetylgalactosaminidase in yeast cells.
  • the cDNA encoding chicken liver ⁇ -N-acetylgalacto- saminidase was subcloned in the EcoRI site of Pichia pastoris expression vector pHIL-Sl (Invitrogen Corp. , San
  • ⁇ -N-acetyl-galactosaminidase enzyme is under the control of the methanol inducible promoter A0X1, and the expressed enzyme is secreted into the culture media via the PhOl signal sequence derived from the pHIL-Sl vector.
  • Pichia pastoris GS-115 was transformed with the plasmid pHO-AZ accordingly to the Invitrogen protocol. Transformants on the plate were screened for high level expression of the enzyme in a filter assay using 2.5 mM of the substrate 5- bromo-4-chloro-3-indolyl- ⁇ D-2-acetylamido-2-deoxylgalacto- pyranoside.
  • a large-scale production of the enzyme was carried out in a 14-L fermentor. After removal of cells from the fermentation culture, the ⁇ -N-acetylgalacto ⁇ saminidase containing supernatant was concentrated and subjected to a strong cation exchange column (Macro-Prep S50, Bio-Rad). After washing off the unbound proteins, a linear NaCl gradient ranging from 50 mM to 350 mM was applied. The SDS-PAGE analysis of the column fractions indicated that the enzyme was homogeneous after the chromatography purification.
  • the recombinant and native ⁇ -N-acetylgalacto- saminidase enzymes were then analyzed on a SDS-PAGE stained with Coomassie blue, and the results are shown in Figure 6A. Based upon the size marker (BioRad, low MW standard) , the recombinant enzyme has a molecular mass of 50 kDA, whereas the native enzyme is 43 kDA. Both enzymes strongly reacted with the anti-sera against the ⁇ -N-acetylgalactosaminidase enzyme.
  • N- glycosidase F specifically cleaves N-linked oligosaccharide chains
  • the recombinant enzyme contains more sugar than the native enzyme as indicated by its greater reduction in size after the enzyme treatment.
  • the recombinant enzyme was then subjected to N- terminal amino acid sequencing on ABI 477A/120A sequencer.

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Abstract

This invention relates to a recombinant enzyme for use in the removal of A antigens from the surface of cells in blood products. Specifically, this invention is directed to a recombinant $(g)a-N-acetylgalactosaminidase enzyme from chicken liver, methods of cloning and expressing said recombinant $(g)a-N-acetylgalactosaminidase enzyme and a method of removing A antigens from the surface of cells in blood products using said recombinant $(g)a-N-acetylgalactosaminidase enzyme.

Description

RECOMBINANT α-N-ACETYLGALACTOSAMINIDASE ENZYME
Statement of Government Interest This invention was made with government support under NMRDC Grant Number N0014-90-J-1638. As such, the government has certain rights in the invention.
FIELD OF THE INVENTION This invention relates to a recombinant enzyme for use in the removal of type A antigens from the surface of cells in blood products, thereby converting certain sub-type A blood products to type 0 blood products and certain type AB blood products to type B blood products. This invention further relates to methods of cloning and expressing said recombinant enzyme. More particularly, this invention is directed to a recombinant chicken liver α-N-acetylgalacto- saminidase enzyme, methods of cloning and expressing said
*recombinant α-N-acetylgalactosaminidase enzyme, and a method of removing type A antigens from the surface of cells in type A and AB blood products using said recombinant α-N-acetylgalactosaminidase enzyme by contacting said enzyme with blood products so as to remove the terminal moiety of the A-antigenic determinant from the surface of cells (for example, erythrocytes) in said blood products, while allowing the structure and function of the cells in the blood products to remain intact. The recombinant α-N-acetylgalactosaminidase enzyme of this invention provides a readily available and cost-efficient enzyme which can be used in the removal of type A antigens from the surface of cells in type A and AB blood products. Treatment of certain sub-type A blood products with the recombinant enzyme of this invention provides a source of cells free of the A antigen, which blood products are thereby rendered useful in transfusion therapy in the same manner of O type blood products. BACKGROUND OF THE INVENTION As used herein, the term "blood products" includes whole blood and cellular components derived from blood, including erythrocytes (red blood cells) and platelets. There are more than thirty blood group (or type) systems, one of the most important of which is the ABO system. This system is based on the presence or absence of antigens A and/or B. These antigens are found on the surface of erythrocytes and on the surface of all endothelial and most epithelial cells as well. The major blood product used for transfusion is erythrocytes, which are red blood cells containing hemoglobin, the principal function of which is the transport of oxygen. Blood of group A contains antigen A on its erythrocytes. Similarly, blood of group B contains antigen B on its erythrocytes. Blood of group AB contains both antigens, and blood of group O contains neither antigen.
The blood group structures are glycoproteins or glycolipids and considerable work has been done to identify the specific structures making up the A and B determinants or antigens. It has been found that the blood group specificity is determined by the nature and linkage of monosaccharides at the ends of the carbohydrate chains. The carbohydrate chains are attached to a peptide or lipid backbone which is embedded in the lipid bi-layer of the membrane of the cells. The most important (immuno-dominant or immuno-determinant) sugar has been found to be
N-acetylgalactosamine for the type A antigen and galactose for the type B antigen. There are three recognized major sub-types of blood type A. These sub-types are known as A1; A intermediate (Aint) and A2. There are both quantitative and qualitative differences which distinguish these three sub-types. Quantitatively, kλ erythrocytes have more antigenic A sites, i.e., terminal N-acetylgalactosamine residues, than Aint erythrocytes which in turn have more antigenic A sites than A2 erythrocytes. Qualitatively, the transferase enzymes responsible for the formation of A antigens differ biochemically from each other in A^ Aint and A2 individuals. Some A antigens found in Ax cells contain dual A antigenic sites. Blood of group A contains antibodies to antigen B.
Conversely, blood of group B contains antibodies to antigen A. Blood of group AB has neither antibody, and blood group 0 has both. A person whose blood contains either (or both) of the anti-A or anti-B antibodies cannot receive a transfusion of blood containing the corresponding incompatible antigen(s) . If a person receives a transfusion of blood of an incompatible group, the blood transfusion recipient's antibodies coat the red blood cells of the transfused incompatible group and cause the transfused red blood cells to agglutinate, or stick together. Transfusion reactions and/or hemolysis (the destruction of red blood cells) may result therefrom.
In order to avoid red blood cell agglutination, transfusion reactions and hemolysis, transfusion blood type is cross-matched against the blood type of the transfusion recipient. For example, a blood type A recipient can be safely transfused with type A blood which contains compatible antigens. Because type 0 blood contains no A or B antigens, it can be transfused into any recipient with any blood type, i.e., recipients with blood types A, B, AB or 0. Thus, type 0 blood is considered "universal", and may be used for all transfusions. Hence, it is desirable for blood banks to maintain large quantities of type 0 blood. However, there is a paucity of blood type 0 donors. Therefore, it is useful to convert types A, B and AB blood to type 0 blood in order to maintain large quantities of universal blood products.
In an attempt to increase the supply of type 0 blood, methods have been developed for converting certain type A, B and AB blood to type 0 blood. For example, U.S. Patent No. 4,609,627 entitled "Enzymatic Conversion of Certain Sub-Type A and AB Erythrocytes" ("the ,627 Patent") , which is incorporated herein by reference, is directed to a process for converting Aint and A2 (including A2B erythrocytes) to erythrocytes of the H antigen type, as well as to compositions of type B erythrocytes which lack A antigens, which compositions, prior to treatment, contained both A and B antigens on the surface of said erythrocytes. The process for converting Aint and A2 erythrocytes to erythrocytes of the H antigen type which is described in the '627 Patent includes the steps of equilibrating certain sub-type A or AB erythrocytes, contacting the equilibrated erythrocytes with purified chicken liver α-N-acetylgalacto¬ saminidase enzyme for a period sufficient to convert the A antigen to the H antigen, removing the enzyme from the erythrocytes and re-equilibrating the erythrocytes. As described in the '627 Patent, α-N-acetylgalactosaminidase obtained from an avian liver (specifically, chicken liver) source was found to have superior activity in respect of enzymatic conversion or cleavage of A antigenic sites.
Prior to the present invention, it was necessary to purify the enzyme from an avian liver source, a process which is time consuming and can be expensive. Hence, a need has arisen to develop an enzyme source which is more readily available. In addition, a need has arisen to develop an enzyme useful in blood product conversion which enzyme is cost-efficient.
A simplified purification process is described in a related application, Serial No. 07/964,756, filed October 22, 1992, entitled "Preparation of Enzyme for Conversion of Sub-Type A and AB Erythrocytes". This process, as described in the related application, utilizes chicken liver as a source of enzyme and, therefore, requires a number of purification steps. Despite this simplified process, it is still desirable to provide a more readily available and controlled source of enzyme, that being cloned and expressed enzyme. This would provide an enzyme source which is more consistent and which is readily purified at less cost and expense, with a still further reduced number of purification steps. Additionally, a recombinant, cloned enzyme allows for specific protein sequence modifications, which can be introduced to generate an enzyme with optimized specific activity, substrate specificity and pH range. α-N-acetylgalactosaminidase enzymes are characterized (and thereby named) by their ability to cleave N-acetylgalactosamine sugar groups. In isolating or identifying these enzymes, their activity is assessed in the laboratory by evaluating cleavage of synthetic substrates which mimic the sugar groups cleaved by the enzymes, with p-nitrophenylglycopyranoside derivatives of the target sugar groups being commonly used. Although very useful in enzyme identification and isolation procedures (the quantitative cleavage of these synthetic substrates can be used to readily distinguish (and thereby identify) enzymes isolated from different sources) , these synthetic substrates are simple structurally and small-sized and mimic only a portion of the natural glycoproteins and glycolipid structures which are of primary concern, those being the A antigens on the surface of cells.
A natural glycolipid substrate, originally isolated from sheep erythrocytes, is the Forsmann antigen (globopentaglycosylceramide) . The Forsmann antigen substrate appropriately mimics the natural A antigen glycolipid structures and is therefore utilized to predict the activity of α-N-acetylgalactosaminidase enzymes against the A antigen substrate. Isolated Forsmann antigen glycolipids have been shown to inhibit hemolysis of sheep red cells by immune rabbit anti-A serum in the presence of serum complement. α-N-acetylgalactosaminindase enzyme has been isolated from a number of sources besides chicken liver (described above) , including bacteria, mollusks, earthworms, and human liver. The human α-N-acetylgalactosaminidase enzyme has been purified, sequenced, cloned and expressed. For example, in "Human α-N-Acetylgalactosaminidase Molecular Cloning, Nucleotide Sequence and Expression of a Full-length cDNA", by Wang et al., in The Journal of Biological Chemistry. Vol. 265, No. 35, pages 21859-21866 (December 15, 1990) , the cDNA encoding human α-N-acetyl¬ galactosaminidase was sequenced. In addition, in "Molecular Cloning of a Full-Length cDNA for Human α-N-Acetylgalacto- saminidase (α-Galactosidase B)", by Tsuji et al., in Biochemical And Biophysical Research Communications. Vol. 163, No. 3, pages 1498-1504 (September 29, 1989), the cDNA encoding human α-N-acetylgalactosaminidase was sequenced. Both the nucleotide sequence and the amino acid sequence of human α-N-acetylgalactosaminidase is published therein. Further, PCT Application No. WO 92/07936 discloses the cloning and expression of the cDNA which encodes human α-N-acetylgalactosaminidase. Although human α-N-acetylgalactosaminidase has been purified, sequenced, cloned and expressed, it is not appropriate for use in removing A antigens from the surface of cells in blood products. In determining whether an enzyme is appropriate for use in removing A antigens from the surface of cells, one must consider the following enzyme characteristics, particularly with respect to the Forsmann antigen substrate: substrate specificity, specific activity or velocity of the substrate cleavage reaction, and pH optimum. Substrate specificity is measured in the Km value, which measures the binding constant or affinity of an enzyme for a particular substrate. The lower a Km value, the more tightly an enzyme binds its substrate. The velocity of an enzyme cleavage reaction is measured in the Vmax, the reaction rate at a saturating concentration of substrate. A higher Vmax indicates a faster cleavage rate. The ratio of these two parameters, Vmax/Km, is a measure of the overall efficiency of an enzyme in reacting with (cleaving) a given substrate. A higher Vmax/Km indicates greater enzyme efficiency. For successful and clinically applicable removal of A antigens from the surface of cells, the enzyme must be sufficiently active at or above a pH at which the cells being treated can be maintained. The procedure described in the '627 patent calls for treatment of cells at or above a pH of 5.6. Therefore, the pH optimum of an appropriate enzyme must still provide reasonable enzyme activity at this pH. These specific characteristics (Vmax/Km, Vmax, Km and pH optimum) are reported for the human a-N-acetylgalactosaminidase enzyme in "Studies on Human Liver a-galactosidases", by Dean et al. in The Journal of Biological Chemistrv. Vol. 254, No. 20, pages 10001-10005 (1979). The Vmax/Km value for the Forsmann antigen of human a-N-acetylgalactosaminidase is 0.46, as compared to a Vmax/Km value of 5.0 for the chicken liver enzyme, indicating an approximately ten-fold difference in efficiency. The Km is lower and the Vmax is higher for the chicken liver enzyme, compared to the human enzyme.
Further, human α-N-acetylgalactosaminidase has a pH optimum for the Forsmann antigen of 3.9, compared to 4.7 for chicken liver α-N-acetylgalactosaminidase. By all of these enzyme characteristics, human α-N-acetylgalactosaminidase enzyme is not suitable for removal of A antigens, particularly when compared to the chicken liver enzyme.
As a result, a need still existed to develop an enzyme which is capable of removing A antigens from the surface of cells in blood products, wherein said enzyme is readily available and cost-efficient.
It is therefore an object of this invention to provide a recombinant enzyme for use in the removal of A antigens from the surface of cells in blood products.
It is another object of this invention to provide a recombinant enzyme for use in the removal of A antigens from the surface of cells in blood products wherein said enzyme is readily available and may be manufactured on a cost-efficient basis.
It is a further object of this invention to provide methods of cloning and expressing a recombinant enzyme useful in the removal of A antigens from the surface of cells in blood products. It is yet another object of this invention to provide a method of removing A antigens from the surface of cells in blood products using a recombinant enzyme.
BRIEF DESCRIPTION OF THE DRAWINGS
The above brief description, as well as further objects and features of the present invention, will be more fully understood by reference to the following detailed description of the presently preferred, albeit illustrative, embodiment of the present invention when taken in conjunction with the accompanying drawing wherein:
Figure 1 represents a diagram of the strategy used to clone and sequence the chicken liver α-N-acetylgalacto¬ saminidase cDNA; Figure 2 represents the nucleic acid sequence and the deduced amino acid sequence of the chicken liver α-N-acetylgalactosaminidase cDNA clone;
Figure 3 represents the expression of chicken liver α-N-acetylgalactosaminidase in bacteria and rabbit reticulocyte lysate aε shown by Western blot;
Figure 4 represents a homology comparison between α-N-acetylgalactosaminidases and a-galactosidases; and
Figure 5 represents the expression of chicken liver α-N-acetylgalactosaminidase in yeast as shown by Western blot.
Figures 6A and 6B represent the determination of the molecular mass of the recombinant α-N-acetylgalacto¬ saminidase enzyme produced by the Pichia pastoris expression system in comparison to the native α-N-acetylgalacto- saminidase enzyme.
Figure 7 represents the results of the N- glycosidase treatment of the recombinant α-N-acetyl¬ galactosaminidase enzyme produced by the Pichia pastoris expression system and the native α-N-acetylgalactosaminidase enzyme. Lanes 1 and 3 correspond to the untreated recombinant and native enzymes, respectively, and lanes 2 and 4 correspond to the N-glycosidase F treated recombinant and native enzymes, respectively. The labels a, b and c on the right side of the blot correspond to the recombinant enzyme, the native enzyme and both deglycosylated enzymes, respectively.
SUMMARY OF THE INVENTION This invention is directed to a recombinant chicken liver α-N-acetylgalactosaminidase enzyme, which enzyme has a molecular weight of about 45 kDa, is immunoreactive with an antibody specific for chicken liver α-N-acetylgalactosaminidase, and also has about 80% amino acid sequence homology with human α-N-acetylgalacto¬ saminidase enzyme. The recombinant chicken liver α-N-acetylgalactosaminidase enzyme of this invention has the amino acid sequence depicted in Figure 2, from amino acid number 1 to amino acid number 406. This invention is further directed to methods of cloning and expressing the recombinant chicken liver α-N-acetylgalactosaminidase enzyme, and to a method of using said enzyme to remove A antigens from the surface of cells in blood products so as to convert said blood products of certain A sub-types to type O, thereby rendering said blood products universal for use in transfusion therapy.
DETAILED DESCRIPTION OF THE INVENTION
This invention is directed to a recombinant enzyme for use in the removal of type A antigens from the surface of cells in blood products, thereby converting certain sub-type A blood products to type 0 blood products and certain sub-type AB blood products to type B blood products. The recombinant chicken liver α-N-acetylgalactosaminidase enzyme of this invention has a molecular weight of about 45 kDa and is immunoreactive with an antibody specific for chicken liver α-N-acetylgalactosaminidase. In addition, the recombinant enzyme of this invention has about 80% amino acid sequence homology with human α-N-acetylgalacto¬ saminidase enzyme. A DNA vector containing a sequence encoding chicken liver α-N-acetylgalactosaminidase was deposited under the Budapest Treaty with the American Type Culture Collection, Rockville, Maryland, on March 17, 1993, tested and found viable on March 22, 1993 and catalogued as ATCC No. 75434.
The recombinant chicken liver α-N- acetylgalactosaminidase enzyme of this invention can be cloned and expressed so that it is readily available for use in the removal of A antigens from the surface of cells in blood products. The enzyme of this invention can be cloned and expressed by screening a chicken liver cDNA library to obtain the cDNA sequence which encodes the chicken liver α-N-acetylgalactosaminidase, sequencing the encoding cDNA once it is determined, cloning the encoding cDNA and expressing α-N-acetylgalactosaminidase from the cloned encoding cDNA. This may be performed by obtaining an amplified human α-N-acetylgalactosaminidase fragment capable of use as a screening probe, screening a chicken liver cDNA library, such as the one described hereinabove, using the amplified human α-N-acetylgalactosaminidase fragment as a probe so as to obtain the cDNA sequence of the chicken liver cDNA library which encodes chicken liver α-N-acetylgalacto¬ saminidase, sequencing the encoding DNA, cloning the encoding DNA and expressing chicken liver α-N-acetylgalacto¬ saminidase enzyme from the cloned encoding cDNA. Alternatively, screening can be performed using antibodies which recognize chicken liver α-N-acetylgalactosaminidase. Methods which are well known to those skilled in the art can be used to construct expression vectors containing the chicken liver α-N-acetylgalactosaminidase coding sequence, with appropriate transcriptional/ translational signals for expression of the enzyme in the corresponding expression systems. Appropriate organisms, cell types and expression systems include: cell-free systems such as a rabbit reticulocyte lysate system, prokaryotic bacteria, such as E. coli, eukaryotic cells, such as yeast, insect cells, mammalian cells (including human hepatocytes or Chinese hamster ovary (CHO) cells) , plant cells or systems, and animal systems including oocytes and transgenic animals. The entire chicken liver α-N-acetylgalacto¬ saminidase coding sequence or functional fragments of functional equivalents thereof may be used to construct the above expression vectors for production of functionally active enzyme in the corresponding expression system. Due to the degeneracy of the DNA code, it is anticipated that other DNA sequences which encode substantially the same amino acid sequence may be used. Additionally, changes to the DNA coding sequence which alter the amino acid sequence of the chicken liver α-N-acetylgalactosaminidase enzyme may be introduced which result in the expression of functionally active enzyme. In particular, amino acid substitutions may be introduced which are based on similarity to the replaced amino acids, particularly with regard to the charge, polarity, hydrophobicity, hydrophilicity, and size of the side chains of the amino acids.
Once a recombinant chicken liver α-N-acetyl¬ galactosaminidase enzyme is cloned and expressed, said enzyme can be used to remove A antigens from the surface of cells in blood products. Methods of utilizing chicken liver α-N-acetylgalactosaminidase to remove A antigens from the surface of erythrocytes can be found in U.S. Patent No. 4,609,627 issued September 2, 1986 to Goldstein, entitled "Enzymatic Conversion of Certain Sub-type A and AB Erythrocytes", which is incorporated herein by reference. Sub-type A antigens can be removed from the surface of erythrocytes by contacting the erythrocytes with the recombinant chicken liver α-N-acetylgalactosaminidase enzyme of this invention for a period of time sufficient to remove the A antigens from the surface of the erythrocytes. EXAMPLE
Isolation and Characterization of the Chicken Liver cDNA Clone
Chicken liver α-N-acetylgalactosaminidase was purified to homogeneity. The enzyme was a glycoprotein with a molecular weight of 80 kDa, and was dissociated into two identical subunits at pH 7.5. Its optimal pH for cleavage of the synthetic p-nitrophenyl-α-N-acetylgalactosaminyl- pyranoside substrate was 3.65 and the activity dropped sharply when the pH was raised above 7. The N-terminal sequence obtained from the purified chicken liver a-N-acetylgalactosaminidase showed a strong homology with the corresponding sequence deduced from the human a-N-acetylgalactosaminidase cDNA clone described in Tsuji et al., and Wang et al.
In order to isolate and characterize the cDNA clone for chicken liver α-N-acetylgalactosaminidase, two -oligonucleotides, corresponding to nucleotides 688 to 705 and 1219 to 1236 of the human α-N-acetylgalactosaminidase sequence published by Wang, et al. were synthesized. Using human placental mRNA (Clontech) as a template, the specific cDNA was made from the downstream (C-terminal) oligonucleotide. Next, a DNA fragment corresponding to human α-N-acetylgalactosaminidase residues from 688 to 1236 was amplified from the cDNA by the hot-start PCR technique. The PCR reaction mixture was preheated at 95°C for 5 minutes and maintained at 80°C while Taq DNA polymerase (Promega) was added to reduce the possible non-specific annealing at lower temperature. 35 cycles of amplification was then carried out as follows: 94°C for 1 minute, 50°C for 2 minutes and 72°C for 3 minutes. The same conditions for PCR were applied in all of the following experiments. The PCR-amplified fragment was then used as a radioactively-labeled probe in the screening of a chicken liver cDNA library (Stratagene) based on homology hybridization. The filters containing the library were hybridized with the probe overnight at 42°C in a solution of 50% formamide, 5XSSPE, 5XDenhardt's, 0.1% SDS and 0.1 mg/ml salmon sperm DNA. The filters were then washed as follows:
1. 3 X SSC + 0.1% SDS, 20 min. room temperature
2. 2 X SSC + 0.1% SDS, 20 min. room temperature 3. 1 X SSC + 0.1% SDS, 20 min. 56°C
4. I X SSC + 0.1% SDS, 20 min. 56°C The filters were autoradiographed overnight at -70°C. The positive clones were picked up for the second-round screening following the same procedure. In total, three consecutive screenings were carried out in order to obtain a well-isolated positive clone.
From approximately one million plaques screened, one positive clone was successfully isolated. The sequencing data indicated that the clone consists of a 1.2 kb 3'-untranslated region and a 0.7 kb coding region which is highly homologous to human α-N-acetylgalacto¬ saminidase. In order to obtain the missing coding sequence, the library was rescreened by using the 1.9 kb cDNA clone as a probe. However, no positive clone was identified by this approach.
The upstream cDNA sequence was then obtained by applying multiple amplification (the nested PCR technique) of a second chicken liver cDNA library (Clontech) . Figure 1 represents a diagram of the strategy used to clone and sequence the chicken liver α-N-acetylgalactosaminidase cDNA. The cDNA encoding chicken liver α-N-acetylgalactosaminidase contained a 1.2 kb coding region (slashed area) and a 1.2 kb 3' untranslated region. The arrows at the bottom of the diagram indicate the sequencing strategy. CL1, CL2 and CL3 are oligonucleotides used as primers for the nested PCR. CL1 and CL2 are located at position 924-941 nt and 736-753 nt, respectively (see Figure 2) . According to the N-terminal sequence of native chicken liver enzyme, the oligonucleotide CL3 [5'-CTGGAGAAC(T)GGA(GC)CTGGCT(CA)CG] was designed taking into account chicken codon usage and "best guess". In the first-round PCR amplification, the whole cDNA library was used as a template in the presence of one specific primer (CL1) (see Figure 1) and one universal primer derived from the library vector (5'-CTGGTAATGGTAG- CGACC) . A small aliquot from the above reaction was directly taken for the second-round amplification with a different set of primers. The primer CL2 had the sequence located upstream of CL1 (Figure 1) and the second primer, CL3, was designed based on the N-terminal amino acid sequence from purified chicken liver α-N-acetylgalacto¬ saminidase (see Figure 1) . A 750 bp fragment was sequenced to eliminate any possible PCR artifacts. Since the 750 bp fragment overlapped with the 1.9 kb clone isolated by the library-screening, the two fragments were linked together by PCR to reconstitute the cDNA encoding chicken liver α-N-acetylgalactosaminidase (Figure 1) . The DNA sequencing was performed according to standard procedure, and the coding region was sequenced in both orientations.
The Cloned DNA Encodes Chicken
Liver a-N-Acetylgalactosaminidase
The authenticity of the cDNA clone was established by co-linearity of deduced amino acid sequences with N-terminal and CNBr-digested peptide sequences from purified chicken liver α-N-acetylgalactosaminidase. Figure 2 represents the nucleic acid sequence and deduced amino acid sequence of the chicken liver α-N-acetylgalactosaminidase cDNA clone. The underlined regions in Figure 2 match sequences obtained from the N-terminus and CNBr-derived fragments of enzyme purified from chicken liver. The first 3 nucleotides, ATG, were added during subcloning to serve as the translational initiation codon for protein expression. The polyadenylation signal (AATAAA) at positions 2299-2304 nt is double-underlined. The boxed sequence indicates potential sites for N-glycosylation. According to the cDNA, the mature protein of 405 amino acids has a molecular mass of about 45 kDa, consistent with that of the purified enzyme estimated by SDS-PAGE. Due to the cloning approach applied, the sequence at the 5' end of the cDNA corresponded to the N-terminal sequence of the mature enzyme isolated from chicken liver.
In order to express the chicken liver a-N-acetylgalactosaminidase in a rabbit reticulocyte lysate, the sequence from 1 to 1260 nucleotides which contained the coding region for chicken liver a-N-acetylgalactosaminidase was subcloned into the vector PCR-II (Invitrogen) in such an orientation that the T7 promoter was located upstream of the insert. Since the N-terminus of the mature protein started with leucine, a translational initiation codon, ATG, was added during the subcloning construction. The construct was then used as a template in a transcription-translation coupled system, TNT system (Promega) , for protein expression according to the procedure recommended by the manufacturer.
In order to produce the recombinant α-N-acetylgalactosaminidase in large quantities in bacteria and purify the enzyme in a single-step fashion, the cDNA was subcloned into the EcoRI site of the pTrcHis vector (Invitrogen) for expression in E. coli. Because of the sequence in the vector, the expressed enzyme contained a polyhistidine-tag in its N-terminus, which permitted one step purification by affinity chromatography from crude cell lysates. Figure 3 represents the expression of chicken liver α-N-acetylgalactosaminidase in bacteria and rabbit reticulocyte lysate as shown by Western blotting. Lane 1 through lane 4 demonstrate the results of expression in a rabbit reticulocyte lysate. The expression was carried out in lysate in the presence of 35S-methionine with (lane 1) or without (lane 2) the expression plasmid. Next, 5 ml of the reaction sample was loaded to a 12% SDS-PAGE. The gel was dried and autoradiographed for 2 hours and a band of an apparent molecular weight of about 45KDa was visualized with the expression plasmid (lane 1, Figure 3) . In order to confirm the authenticity of the expressed protein, a Western blot was performed using a polyclonal antibody raised against α-N-acetylgalactosaminidase purified from chicken liver. Using non-labelled methionine instead, the same expression reaction was performed for a Western blot (Promega) as shown in lanes 3 and 4, with and without the expression plasmid, respectively. As indicated in Figure 3, the antibody specifically recognized a band from the reaction with expression plasmid (lane 3) , but not in the control (lane 4) . Lane 5 shows the protein expressed in bacteria and recognized by the same antibody on Western blot. Lane 6 shows the α-N-acetylgalactosaminidase purified from chicken liver as a positive control. Molecular weight size marker (m) is indicated on the left. Hence, it was confirmed that the isolated cDNA clone codes for the chicken liver α-N-acetylgalactosaminidase.
Comparison of the Cloned Chicken Liver Sequence with other Enzyme Sequences
The chicken liver α-N-acetylgalactosaminidase * sequence was compared with published sequences of other α-N-acetylgalactosaminidases and α-galactosidases which cleave α-galactose sugar groups. Figure 4 shows a homology comparison between various α-N-acetylgalactosaminidases and α-galactosidases. Alignment was carried out using both the computer program PROSIS (Hitachi Software Engineering Corp., Ltd.) and manual arrangement. The amino acid sequences were deduced from cDNAs. Sequences I and II are of α-N-acetylgalactosaminidases from chicken liver and human placenta, respectively. Sequences III, IV, V and VI represent α-galactosidase from human, yeast, Cvamopsis tetragonoloba and Aspergillus niger. respectively. Sequences IV and VI are truncated at the C-terminus, as indicated by **. Identical or conservatively substituted amino acid residues (five out of six or more) among the aligned protein sequences are boxed. The numbers above the sequences indicate the relative position of each peptide sequence.
The deduced amino acid sequence from chicken liver α-N-acetylgalactosaminidase cDNA shows approximately 80% homology with the human α-N-acetylgalactosaminidase as determined by PROSIS. This homology indicates the relatedness of the human and chicken liver enzymes, despite the differences in the specific characteristics of the enzymes, particularly with regard to cleavage of the Forsmann antigen, as has already been described. Also, polyclonal antibodies raised against chicken liver α-N-acetylgalactosaminidase enzyme do not cross react with the human enzyme. The specific amino acids responsible for these differences remain to be elucidated.
Yamachi et al. (1990) reported that a human α-N-acetylgalactosaminidase cDNA with an insertion of 70bp at the position corresponding to number 376 in Figure 4 was not enzymatically active in a transient expression study in COS cells. The data suggests that the open reading frame shift caused by this insertion in the C-terminal portion of the molecule is responsible for the loss of enzymatic activity, indicating that amino acids in the C-terminal region may be essential for α-N-acetylgalactosaminidase enzyme activity.
By sequence similarity searching (BLAST) (Altschul et al. 1990) of available protein databases followed by sequence alignment using the PROSIS computer program and manual arrangement, it was found that α-N-acetylgalacto- saminidase is highly homologous to α-galactosidases from human, yeast, cya opsis tetragonoloba and aspergillus niger (ranging from 55% to 68% at the amino acid level) . The extent of the amino acid sequence homology, as shown in Figure 4, suggests that these two functionally specific glycosidases might have evolved from a common ancestral gene. Considering the high degree of similarities and the nature of their substrates it is possible that the two exoglycosidases share a similar catalytic mechanism and the critical amino acid residues involved in both active sites are well conserved. The addition of chicken liver α-N-acetylgalactosaminidase cDNA to the family provides further insight into regions of the molecule which are important for the substrate binding specificity and enzymatic activity. Given the availability of cloned enzymes from a number of sources, the active site and catalytic mechanisms of a-N-acetylgalactosaminidase and α-galactosidase enzymes may now be studied by means of cDNA deletion and site-directed mutagenesis.
Expression of Active Chicken Liver α-N-acetylgalactosaminidase in Yeast
The first 48 nucleotides of human α-N-acetyl¬ galactosaminidase cDNA (Wang, et al. 1990) which correspond to the signal peptide sequence, were linked to the cloned chicken liver α-N-acetylgalactosaminidase coding region by PCR. The PCR amplified product was subcloned directly into the vector PCR-II (Invitrogen) . Two EcoRI sites flanking the insert were used to subclone the entire α-N-acetyl¬ galactosaminidase cDNA into the yeast expression vector pYES2 (Invitrogen) in such an orientation that the GAL 1 promoter was located upstream of the insert. The GAL 1 promoter provides expression of the inserted cDNA clone under galactose inducing growth conditions in yeast.
The yeast vector constructs were transformed into the yeast strain, INVSCI (Invitrogen) using standard procedures. To confirm the expression of the chicken liver α-N-acetylgalactosaminidase in yeast, the total proteins from cell extract and culture supernatant were prepared and separated by 12% SDS-PAGE and a Western blot performed (by standard conditions) using the polyclonal antibody raised against purified chicken liver α-N-acetylgalactosaminidase.
The transformed yeast cells were grown in medium without uracil (Bio 101, Inc.). After 0.2% galactose induction, the cells were centrifuged and protein extracts were prepared using glass bead disruption. The secreted proteins in the culture supernatant were concentrated with a Centricon-30
(Amicon Division, W.R. Grace & Co.). The Western blot results are depicted in Figure 5.
Lanes 1 and 8 of Figure 5 show the α-N-acetylgalactosaminidase purified from chicken liver. Lane 2 through lane 4 are cell extracts from the yeast transformed with three different pYES2 constructs: the vector alone (lane 2) , chicken liver α-N-acetylgalacto¬ saminidase cDNA coding region (lane 3) , and the coding region plus signal sequence (lane 4) . Lane 5 is the culture supernatant from transformed yeast used in Lane 4. Lane 7 shows the molecular weight standard. As shown in Figure 5, while the protein without signal peptide was expressed within yeast cells (lane 3) , the protein with a signal peptide sequence was predominantly secreted into the media (lane 5) . The larger molecular weight of the secreted protein observed on the Western blot was presumably caused by overglycosylation, as was observed for the expression of guar α-galactosidase in yeast (Fellinger, et al. 1991) . To purify the expressed α-N-acetylgalacto¬ saminidase, concentrated culture supernatant was applied to an affinity column containing aminocaproylgalactosylamine agarose. After washing the column, the bound fraction was eluted with buffer containing 50mM N-acetylgalactosamine. This eluate contains expressed α-N-acetylgalactosaminidase of similar molecular weight to that of the enzyme purified from chicken liver, as indicated in lane 6 in Figure 5.
The expressed enzyme eluted from the column demonstrates activity toward the synthetic substrate p-nitrophenyl-α-N-acetylgalactosaminylpyranoside at pH 3.6. Heavily glycosylated enzyme did not bind to the affinity column and showed no activity against synthetic substrate. All the data taken together demonstrate production, secretion and purification of enzymatically active chicken liver α-N-acetylgalactosaminidase in yeast cells.
Expression of Chicken Liver α-N-acetylgalactosaminidase in Pichia pastoris
The cDNA encoding chicken liver α-N-acetylgalacto- saminidase was subcloned in the EcoRI site of Pichia pastoris expression vector pHIL-Sl (Invitrogen Corp. , San
Diego, CA) generating the plasmid pHO-AZ. The expression of α-N-acetyl-galactosaminidase enzyme is under the control of the methanol inducible promoter A0X1, and the expressed enzyme is secreted into the culture media via the PhOl signal sequence derived from the pHIL-Sl vector. Pichia pastoris (GS-115) was transformed with the plasmid pHO-AZ accordingly to the Invitrogen protocol. Transformants on the plate were screened for high level expression of the enzyme in a filter assay using 2.5 mM of the substrate 5- bromo-4-chloro-3-indolyl-αD-2-acetylamido-2-deoxylgalacto- pyranoside. A large-scale production of the enzyme was carried out in a 14-L fermentor. After removal of cells from the fermentation culture, the α-N-acetylgalacto¬ saminidase containing supernatant was concentrated and subjected to a strong cation exchange column (Macro-Prep S50, Bio-Rad). After washing off the unbound proteins, a linear NaCl gradient ranging from 50 mM to 350 mM was applied. The SDS-PAGE analysis of the column fractions indicated that the enzyme was homogeneous after the chromatography purification.
The recombinant and native α-N-acetylgalacto- saminidase enzymes were then analyzed on a SDS-PAGE stained with Coomassie blue, and the results are shown in Figure 6A. Based upon the size marker (BioRad, low MW standard) , the recombinant enzyme has a molecular mass of 50 kDA, whereas the native enzyme is 43 kDA. Both enzymes strongly reacted with the anti-sera against the α-N-acetylgalactosaminidase enzyme.
The recombinant and native α-N-acetylgalacto¬ saminidase enzymes (10 μg each) , after denaturation, were then treated with 0.4 units of N-glycosidase F. After incubation at 37°C overnight the samples were analyzed on a SDS gel for Western blot using an antibody against the purified enzyme. As shown in Figure 7, both the recombinant and the native enzymes migrated faster after the N- glycosidase F treatment (lanes 2 and 4) in comparison with untreated controls (lanes 1 and 3, respectively). Since N- glycosidase F specifically cleaves N-linked oligosaccharide chains, the data suggested that the recombinant enzyme, as well as the native enzyme, are both glycosylated. However, the recombinant enzyme contains more sugar than the native enzyme as indicated by its greater reduction in size after the enzyme treatment. There are three potential N- glycosylation sites based on the cDNA sequence coding for the enzyme, although it is not clear which sites are used for glycosylation.
The recombinant enzyme was then subjected to N- terminal amino acid sequencing on ABI 477A/120A sequencer. The data indicated that the pHOI secretion signal was cleaved correctly, generating the recombinant enzyme with Arg as the N-terminus. Therefore, in comparison with its native counterpart, the recombinant enzyme has four extra residues in its N-terminus which were generated during the construction of the plasmid, pHO-AZ.
The specific activity, optimal pH, Vmaχ and K,,, for both the recombinant and native enzymes were determined, and the results are as follows:
Spec. Act. 1 Vmax Km (U/mq) Optimal pH (mM) (mM)
Recomb. enzyme 51.2 3.65 60.9 0.827
Native enzyme 56.4 3.65 75.7 0.798
1 Both enzymes are equally stable at 37°C. At the end of 5 hours of incubation over 95% of activity remained.
Although the invention herein has been described with reference to particular embodiments, it is to be understood that these embodiments are merely illustrative of various aspects of the invention. Thus, it is to be understood that numerous modifications may be made in the illustrative embodiments and other arrangements may be devised without departing from the spirit and scope of the invention. SEQUENCE LISTING
(1) GENERAL INFORMATION
(i) APPLICANT: NEW YORK BLOOD CENTER, INC.
(ii) TITLE OF INVENTION: RECOMBINANT
ALPHA-N-ACETYLGALACTOSAMINIDASE ENZYME
(iii) NUMBER OF SEQUENCES: 7
(iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: AMSTER, ROTHSTEIN & EBENSTEIN
(B) STREET: 90 PARK AVENUE
(C) CITY: NEW YORK
(D) STATE: NEW YORK
(E) COUNTRY: U.S.A.
(F) ZIP: 10016
(V) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: 3.5 INCH 1.44 Mb STORAGE DISKETTE
(B) COMPUTER: IBM PC COMPATIBLE
(C) OPERATING SYSTEM: MS-DOS
(D) SOFTWARE: ASCII
- (vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER: NOT YET ASSIGNED
(B) FILING DATE: NOT YET ASSIGNED
(viii) ATTORNEY/AGENT INFORMATION:
(A) NAME: CRAIG J. ARNOLD
(B) REGISTRATION NUMBER: 34,287
(C) REFERENCE/DOCKET NUMBER: 63475/99
(ix) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: (212) 697-5995
(B) TELEFAX: (212) 286-0854 or 286-0082
(C) TELEX: TWX 710-581-4766
(2) INFORMATION FOR SEQ ID NO: 1
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 2319
(B) TYPE: NUCLEIC ACID
(C) STRANDEDNESS: DOUBLE
(D) TOPOLOGY: LINEAR
(ii) MOLECULE TYPE:
(A) DESCRIPTION: OLIGONUCLEOTIDE
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: YES (vi) ORIGINAL SOURCE:
(A) ORGANISM: CHICKEN LIVER
(B) INDIVIDUAL ISOLATE: ALPHA-N- ACETYLGALACTOSAMINIDASE
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1
ATG CTG GAG AAC GGG CTG GCG CGG ACC CCG CCC ATG GGC TGG TTG GCC 48 Met Leu Glu Asn Gly Leu Ala Arg Thr Pro Pro Met Gly Trp Leu Ala
TGG GAG CGG TTC CGC TGC AAC GTG AAC TGC CGG GAG GAC CCC CGC CAG 96 Trp Glu Arg Phe Arg Cys Asn Val Asn Cys Arg Glu Asp Pro Arg Gin
TGC ATC AGT GAG ATG CTC TTC ATG GAG ATG GCA GAC CGA ATA GCA GAG 144 Cys lie Ser Glu Met Leu Phe Met Glu Met Ala Asp Arg lie Ala Glu
GAC GGC TGG AGG GAG CTG GGC TAC AAG TAC ATC AAT ATC GAT GAC TGC 192 Asp Gly Trp Arg Glu Leu Gly Tyr Lys Tyr lie Asn lie Asp Asp Cys
TGG GCC GCC AAG CAG CGT GAC ACT GAG GGG CGG CTG GTG CCT GAC CCC 240 Trp Ala Ala Lys Gin Arg Asp Thr Glu Gly Arg Leu Val Pro Asp Pro
GAG AGG TTC CCC CGG GGC ATT AAG GCC TTG GCT GAC TAC GTT CAT GCC 288 Glu Arg Phe Pro Arg Gly lie Lys Ala Leu Ala Asp Tyr Val His Ala
CGA GGC TTG AAG CTG GGC ATT TAT GGC GAC CTG GGC AGA CTC ACC TGT 336 Arg Gly Leu Lys Leu Gly lie Tyr Gly Asp Leu Gly Arg Leu Thr Cys
GGA GGC TAC CCA GGC ACC ACG CTG GAC CGT GTG GAG CAG GAC GCA CAG 384 Gly Gly Tyr Pro Gly Thr Thr Leu Asp Arg Val Glu Gin Asp Ala Gin
ACC TTC GCT GAG TGG GGT GTG GAC ATG CTG AAG CTA GAT GGG TGC TAC 432 Thr Phe Ala Glu Trp Gly Val Asp Met Leu Lys Leu Asp Gly Cys Tyr
TCA TCG GGG AAG GAG CAG GCA CAG GGC TAC CCA CAA ATG GCA AGG GCC 480 Ser Ser Gly Lys Glu Gin Ala Gin Gly Tyr Pro Gin Met Ala Arg Ala
TTG AAC GCC ACT GGC CGC CCC ATC GTC TAC TCC TGC AGC TGG CCA GCC 528 Leu Asn Ala Thr Gly Arg Pro lie Val Tyr Ser Cys Ser Trp Pro Ala
TAC CAG GGG GGG CTG CCT CCC AAG GTG AAC TAC ACT CTC CTG GGT GAG 576 Tyr Gin Gly Gly Leu Pro Pro Lys Val Asn Tyr Thr Leu Leu Gly Glu
ATC TGC AAC CTG TGG CGG AAC TAC GAT GAC ATC CAG GAC TCA TGG GAC 624 lie Cys Asn Leu Trp Arg Asn Tyr Asp Asp lie Gin Asp Ser Trp Asp
AGC GTG CTT TCC ATC GTG GAC TGG TTC TTC ACA AAC CAG GAT GTG CTG 672 Ser Val Leu Ser lie Val Asp Trp Phe Phe Thr Asn Gin Asp Val Leu
CAG CCG TTT GCT GGC CCT GGC CAC TGG AAT GAC CCA GAC ATG CTC ATC 720 Gin Pro Phe Ala Gly Pro Gly His Trp Asn Asp Pro Asp Met Leu lie
ATT GGA AAT TTC GGT CTC AGC TAT GAG CAG TCA CGT TCC CAA ATG GCC 768 lie Gly Asn Phe Gly Leu Ser Tyr Glu Gin Ser Arg Ser Gin Met Ala TTG TGG ACC ATT ATG GCA GCT CCA CTC CTC ATG TCC ACC GAC CTG CGC 816 Leu Trp Thr He Met Ala Ala Pro Leu Leu Met Ser Thr Asp Leu Arg
ACT ATC TCG CCG AGT GCC AAG AAG ATT CTG CAG AAC CGC CTG ATG ATC 864 Thr He Ser Pro Ser Ala Lys Lys He Leu Gin Asn Arg Leu Met He
CAG ATA AAC CAG GAC CCC TTG GGA ATC CAG GGG CGC AGG ATC ATC AAG 912 Gin He Asn Gin Asp Pro Leu Gly He Gin Gly Arg Arg He He Lys
GAG GGA TCC CAC ATT GAG GTG TTC CTG CGC CCG CTG TCA CAG GCT GCC 960 Glu Gly Ser His He Glu Val Phe Leu Arg Pro Leu Ser Gin Ala Ala
AGT GCC CTG GTC TTC TTC AGC CGG AGG ACA GAC ATG CCC TTC CGC TAC 1008 Ser Ala Leu Val Phe Phe Ser Arg Arg Thr Asp Met Pro Phe Arg Tyr
ACC ACC AGT CTT GCC AAG CTT GGC TTC CCC ATG GGA GCT GCA TAT GAG 1056 Thr Thr Ser Leu Ala Lys Leu Gly Phe Pro Met Gly Ala Ala Tyr Glu
GTG CAA GAC GTG TAC AGT GGG AAG ATC ATC AGT GGC CTG AAG ACA GGA 1104 Val Gin Asp Val Tyr Ser Gly Lys He He Ser Gly Leu Lys Thr Gly
GAC AAC TTC ACA GTG ATC ATC AAC CCC TCA GGG GTG GTG ATG TGG TAC 1152 Asp Asn Phe Thr Val He He Asn Pro Ser Gly Val Val Met Trp Tyr
CTG TGT CCC AAA GCA CTG CTC ATC CAG CAG CAA GCT CCT GGG GGG CCC 1200 Leu Cys Pro Lys Ala Leu Leu He Gin Gin Gin Ala Pro Gly Gly Pro
TCG CGC CTG CCC CTT CTG TGA GGC CCA TGA TTG GGA GCC CTG GGA TAC 1248 Sef Arg Leu Pro Leu Leu
ATC TCA CCG CTG CTC AAG TGC CTT CTT CTG GTG TGG CTG GGG GAG GAC 1296
ATG CAG CTT GCT CCT CTG GCA CCA CCT GAT GAT TTC TAC TCA TTC CAC 1344
GTG AAG CAG GAC TTC TTG TTA CTC CCT CCT GAG AGC ATG CAA AGC GCT 1392
CTG AGG TCC TCC TGT GGA AGA GGA GTG TTC CCA GTG ACC ATC CTT TAG 1440
GAC CAG ATG TGG TCA CCT TTT TTC CTT TGC TTG GCT TAG GAC AAA GGG 1488
CTG TCC ACA GGC TGC ACC CCT CTT CCC AGG CAC CAT CCC CAG ACC AGG 1536
AGC TCC TGG GGC CAG GCT GTC TCT GTC TGG CAG CAG GAT CAG CAG GTA 1584
ACA CCA CTA CAG TGT AGT CCG CAC ATA ATG AAA AAG AAA TCT AAA CAA 1632
AAC GTG TGC CAG TAG TGT ACT GAA CCC GCT CTG GTT ACA GCA GAG CAA 1680
AAC CTG AGT TGT CCA TGC ACA ATC CCA GTA TCC TCA CTG TGG TGT TAG 1728
CAT GAA AAA TTG CAG TCA CAG TGC ATT GTG CAC GAG TGG TGT CTG GAA 1776
GAT GCT GAT GCT TGT TCG TGG TGG TCT TAA GGT GGG AGA TGC TCA TGG 1824
GTG CTG GCC AAG TTG CAT CTC AAT CTT GTG AGG CTG AAC CTT CCA GCA 1872 TTT CTC AGG GAA AGG CTC TTC CTT TTA AAG GCA GCC TGC ACA AAT AGA 1920
AGG GGC TCA GAA GGA CGC ACG AGG AGG GGC TCA GGT GGG CCG TGC TCC 1968
CCT GAC CAC CCC AAG AGG GGT CAA CTA CTC ACC AAA ATC TAC CCC TTT 2016
CAA GGC CAG GTC AGC CCA GGG AGA CGC ACC CAA GGT TAA ACC TCA AAA 2064
CAG GAA ATC ACC CTA TTT TAA ATT AGT GAG AAA TTG AAC TTC CCC ATT 2112
CTA TTC AGA TGA GGG CTA GAA GCC CAC TCT CCT TAG AAG GCA CGT GGT 2160
GGA TTC CTG CCC CTT GCA GAG ACA TTG TGG TCT GAA GCA AGA TGC TGA 2208
ATG TGA TCT TTG CAG CGC TGG AAA TGA CAT GTC TGT TTC ATG CTT GTG 2256
TGG GAG ATG GCT TTG TTT TTG TGA TTT TGA CAA TTT AAC TGA AAT AAA 2304
AGG GAA GCA GAG GGG 2319
(3) INFORMATION FOR SEQ ID NO: 2
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 406
(B) TYPE: AMINO ACID
* (ϋ) MOLECULE TYPE:
(A) DESCRIPTION: PROTEIN
(iii) HYPOTHETICAL: NO
(vi) ORIGINAL SOURCE:
(A) ORGANISM: CHICKEN LIVER
(B) INDIVIDUAL ISOLATE: ALPHA-N- ACETYLGALACTOSAMINIDASE
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2
Met Leu Glu Asn Gly Leu Ala Arg Thr Pro Pro Met Gly Trp Leu Ala 16
Trp Glu Arg Phe Arg Cys Asn Val Asn Cys Arg Glu Asp Pro Arg Gin 32
Cys He Ser Glu Met Leu Phe Met Glu Met Ala Asp Arg He Ala Glu 48
Asp Gly Trp Arg Glu Leu Gly Tyr Lys Tyr He Asn He Asp Asp Cys 64
Trp Ala Ala Lys Gin Arg Asp Thr Glu Gly Arg Leu Val Pro Asp Pro 80
Glu Arg Phe Pro Arg Gly He Lys Ala Leu Ala Asp Tyr Val His Ala 96
Arg Gly Leu Lys Leu Gly He Tyr Gly Aεp Leu Gly Arg Leu Thr Cys 112
Gly Gly Tyr Pro Gly Thr Thr Leu Asp Arg Val Glu Gin Asp Ala Gin 128 Thr Phe Ala Glu Trp Gly Val Asp Met Leu Lys Leu Asp Gly Cys Tyr 144
Ser Ser Gly Lys Glu Gin Ala Gin Gly Tyr Pro Gin Met Ala Arg Ala 160
Leu Asn Ala Thr Gly Arg Pro He Val Tyr Ser Cys Ser Trp Pro Ala 176
Tyr Gin Gly Gly Leu Pro Pro Lys Val Asn Tyr Thr Leu Leu Gly Glu 192
He Cys Asn Leu Trp Arg Asn Tyr Asp Asp He Gin Asp Ser Trp Asp 208
Ser Val Leu Ser He Val Asp Trp Phe Phe Thr Asn Gin Asp Val Leu 224
Gin Pro Phe Ala Gly Pro Gly His Trp Asn Asp Pro Asp Met Leu He 240
He Gly Asn Phe Gly Leu Ser Tyr Glu Gin Ser Arg Ser Gin Met Ala 256
Leu Trp Thr He Met Ala Ala Pro Leu Leu Met Ser Thr Asp Leu Arg 272
Thr He Ser Pro Ser Ala Lys Lys He Leu Gin Asn Arg Leu Met He 288
Gin He Asn Gin Asp Pro Leu Gly He Gin Gly Arg Arg He He Lys 304
Glu Gly Ser His He Glu Val Phe Leu Arg Pro Leu Ser Gin Ala Ala 320
Ser Ala Leu Val Phe Phe Ser Arg Arg Thr Asp Met Pro Phe Arg Tyr 336
Thr Thr Ser Leu Ala Lys Leu Gly Phe Pro Met Gly Ala Ala Tyr Glu 352
Val- Gin Asp Val Tyr Ser Gly Lys He He Ser Gly Leu Lys Thr Gly 368
Asp Asn Phe Thr He Val He Asn Pro Ser Gly Val Val Met Trp Tyr 384
Leu Cys Pro Lys Ala Leu Leu He Gin Gin Gin Ala Pro Gly Gly Pro 400
Ser Arg Leu Pro Leu Leu 406
(4) INFORMATION FOR SEQ ID NO: 3
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 411
(B) TYPE: AMINO ACID
(ii) MOLECULE TYPE:
(A) DESCRIPTION: PROTEIN
(iii) HYPOTHETICAL: NO
(Vi) ORIGINAL SOURCE:
(A) ORGANISM: HUMAN
(B) INDIVIDUAL ISOLATE: ALPHA-N- ACETYLGALACTOSAMINIDASE
(Xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3 Met Leu Leu Lys Thr Val Leu Leu Leu Gly His Val Ala Gin Val Leu 16
Met Leu Asp Asn Gly Leu Leu Gin Thr Pro Pro Met Gly Trp Leu Ala 32
Trp Glu Arg Phe Arg Cys Asn He Asn Cys Asp Glu Asp Pro Lys Asn 48
Cys He Ser Glu Gin Leu Phe Met Glu Met Ala Asp Arg Met Ala Gin 64
Aεp Gly Trp Arg Asp Met Gly Tyr Thr Tyr Leu Asn He Asp Asp Cys 80
Trp He Gly Gly Arg Asp Ala Ser Gly Arg Leu Met Pro Asp Pro Lys 96
Arg Phe Pro His Gly He Pro Phe Leu Ala Asp Tyr Val His Ser Leu 112
Gly Leu Lys Leu Gly He Tyr Ala Asp Met Gly Asn Phe Thr Cys Met 128
Gly Tyr Pro Gly Thr Thr Leu Asp Lys Val Val Gin Asp Ala Gin Thr 144
Phe Ala Glu Trp Lys Val Asp Met Leu Lys Leu Asp Gly Cys Phe Ser 160
Thr Pro Glu Glu Arg Ala Gin Gly Tyr Pro Lys Met Ala Ala Ala Leu 176
Asn Ala Thr Gly Arg Pro He Ala Phe Ser Cys Ser Trp Pro Ala Tyr 192
Glu Gly Gly Leu Pro Pro Arg Val Asn Tyr Ser Leu Leu Ala Asp He 208
Cys Asn Leu Trp Arg Asn Tyr Asp Asp He Gin Asp Ser Trp Trp Ser 224
Va*l Leu Ser He Leu Asn Trp Phe Val Glu His Gin Asp He Leu Gin 240
Pro Val Ala Gly Pro Gly His Trp Asn Asp Pro Asp Met Leu Leu He 256
Gly Asn Phe Gly Leu Ser Leu Glu Gin Ser Arg Ala Gin Met Ala Leu 272
Trp Thr Val Leu Ala Ala Pro Leu Leu Met Ser Thr Asp Leu Arg Thr 288
He Ser Ala Gin Asn Met Asp He Leu Gin Asn Pro Leu Met He Lys 304
He Asn Gin Asp Pro Leu Gly He Gin Gly Arg Arg He His Lys Glu 320
Lys Ser Leu He Glu Val Tyr Met Arg Pro Leu Ser Asn Lys Ala Ser 336
Ala Leu Val Phe Phe Ser Cys Arg Thr Asp Met Pro Tyr Arg Tyr His 352
Ser Ser Leu Gly Gin Leu Asn Phe Thr Gly Ser He Val Tyr Glu Ala 368
Gin Asp Val Tyr Ser Gly Asp He He Ser Gly Leu Arg Asp Glu Thr 384
Asn Phe Thr He Val He Asn Pro Ser Gly Val Val Met Trp Tyr Leu 400
Tyr Pro He Lys Asn Leu Glu Met Ser Gin Gin 411 (5) INFORMATION FOR SEQ ID NO: 4
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 429
(B) TYPE: AMINO ACID
(ii) MOLECULE TYPE:
(A) DESCRIPTION: PROTEIN
(iii) HYPOTHETICAL: NO
(vi) ORIGINAL SOURCE:
(A) ORGANISM: HUMAN
(B) INDIVIDUAL ISOLATE: ALPHA-GALACTOSIDASE
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4
Met Gin Leu Arg Asn Pro Glu Leu His Leu Gly Cys Ala Leu Ala Leu 16
Arg Phe Leu Ala Leu Val Ser Trp Asp He Pro Gly Ala Arg Ala Leu 32
Asp Asn Gly Leu Ala Arg Thr Pro Thr Met Gly Trp Leu His Trp Glu 48
Arg Phe Met Cys Asn Leu Asp Cys Gin Glu Glu Pro Asp Ser Cys He 64
Ser Glu Lys Leu Phe Met Glu Met Ala Glu Leu Met Val Ser Glu Gly 80
Trp Lys Asp Ala Gly Tyr Glu Tyr Leu Cys He Asp Asp Cys Trp Met 96
Ala Pro Gin Arg Asp Ser Glu Gly Arg Leu Gin Ala Asp Pro Gin Arg 112
Phe Pro His Gly He Arg Gin Leu Ala Asn Tyr Val His Ser Lys Gly 128
Leu Lys Leu Gly He Tyr Ala Asp Val Gly Asn Lys Thr Cys Ala Gly 144
Phe Pro Gly Ser Phe Gly Tyr Tyr Asp He Asp Ala Gin Thr Phe Ala 160
Asp Trp Gly Val Asp Leu Leu Lys Phe Asp Gly Cys Tyr Cys Asp Ser 176
Leu Glu Asn Leu Ala Asp Gly Tyr Lys His Met Ser Leu Ala Leu Asn 192
Arg Thr Gly Arg Ser He Val Tyr Ser Cys Glu Trp Pro Leu Tyr Met 208
Trp Pro Phe Gin Lys Pro Asn Tyr Thr Glu He Arg Gin Tyr Cys Asn 224
His Trp Arg Asn Phe Ala Asp He Asp Asp Ser Trp Lys Ser He Lys 240
Ser He Leu Asp Trp Thr Ser Phe Asn Gin Glu Arg He Val Asp Val 256
Ala Gly Pro Gly Gly Trp Asn Asp Pro Asp Met Leu He Val Gly Asn 272
Phe Gly Leu Ser Trp Asn Gin Gin Val Thr Gin Met Ala Leu Trp Ala 288
He Met Ala Ala Pro Leu Phe Met Ser Asn Asp Leu Arg His He Ser 304
Pro Gin Ala Lys Ala Leu Leu Gin Asp Lys Asp He Val Ala He Asn 320 Gln Asp Pro Leu Gly Lys Gin Gly Tyr Gin Leu Arg Gin Gly Asp Asn 336
Phe Glu Val Trp Glu Arg Pro Leu Ser Gly Leu Ala Trp Ala Val Ala 352
Met He Asn Arg Gin Glu He Gly Gly Pro Arg Ser Tyr Thr He Ala 368
Val Ala Ser Leu Gly Lys Gly Val Ala Cys Asn Pro Ala Cys Phe He 384
Thr Gin Leu Leu Pro Val Lys Arg Lys Leu Gly Phe Tyr Glu Trp Thr 400
Ser Arg Leu Arg Ser His He Asn Pro Thr Gly Thr Val Leu Leu Gin 416
Leu Glu Asn Thr Met Gin Met Ser Leu Lys Asp Leu Leu 429
(6) INFORMATION FOR SEQ ID NO: 5
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 438
(B) TYPE: AMINO ACID
(ii) MOLECULE TYPE:
(A) DESCRIPTION: PROTEIN
(iii) HYPOTHETICAL: NO
(vi) ORIGINAL SOURCE:
(A) ORGANISM: YEAST SACCHAROMYCES CERVISIAE
(B) INDIVIDUAL ISOLATE: ALPHA-GALACTOSIDASE
(Xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5
Met Phe Ala Phe Tyr Phe Leu Thr Ala Cys He Ser Leu Lys Gly Val 16
Phe Gly Val Ser Pro Ser Tyr Asn Gly Leu Gly Leu Thr Pro Gin Met 32
Gly Trp Asp Asn Trp Asn Thr Phe Ala Cys Asp Val Ser Glu Gin Leu 48
Leu Leu Asp Thr Ala Asp Arg He Ser Asp Leu Gly Leu Lys Asp Met 64
Gly Tyr Lys Tyr He He Leu Asp Asp Cys Trp Ser Ser Gly Arg Asp 80
Ser Asp Gly Phe Leu Val Ala Asp Glu Gin Lys Phe Pro Asn Gly Met 96
Gly His Val Ala Asp His Leu His Asn Asn Ser Phe Leu Phe Gly Met 112
Tyr Ser Ser Ala Gly Glu Tyr Thr Cys Ala Gly Tyr Pro Gly Ser Leu 128
Gly Arg Glu Glu Glu Asp Ala Gin Phe Phe Ala Asn Asn Arg Val Asp 144
Tyr Leu Lys Tyr Asp Asn Cys Tyr Asn Lys Gly Gin Phe Gly Thr Pro 160
Glu He Ser Tyr His Arg Tyr Lys Ala Met Ser Asp Ala Leu Asn Lys 176 Thr Gly Arg Pro He Phe Tyr ser Leu Cys Asn Trp Gly Gin Asp Leu 192
Thr Phe Tyr Trp Gly Ser Gly He Ala Asn Ser Trp Arg Met Ser Gly 208
Asp Val Thr Ala Glu Phe Thr Arg Pro Asp Ser Arg Cys Pro Cys Asp 224
Gly Asp Glu Tyr Asp Cys Lys Tyr Ala Gly Phe His Cys Ser He Met 240
Asn He Leu Asn Lys Ala Ala Pro Met Gly Gin Asn Ala Gly Val Gly 256
Gly Trp Asn Asp Leu Asp Asn Leu Glu Val Gly Val Gly Asn Leu Thr 272
Asp Asp Glu Glu Lys Ala His Phe Ser Met Trp Ala Met Val Lys Ser 288
Pro Leu He He Gly Ala Asn Val Asn Asn Leu Lys Ala Ser Ser Tyr 304
Ser He Tyr Ser Gin Ala Ser He Val Ala He Asn Gin Asp Ser Asn 320
Gly He Pro Ala Thr Arg Val Trp Arg Tyr Tyr Val Ser Asp Thr Asp 336
Glu Tyr Gly Gin Gly Glu He Gin Met Trp Ser Gly Pro Leu Asp Asn 352
Gly Asp Gin Val Val Ala Leu Leu Asn Gly Gly Ser Val Ser Arg Pro 368
Met Asn Thr Thr Leu Glu Glu He Phe Phe Asp Ser Asn Leu Gly Ser 384
Lys Lys Leu Thr Ser Thr Trp Asp He Tyr Asp Leu Trp Ala Asn Arg 400
Val Asp Asn Ser Thr Ala Ser Ala He Leu Gly Arg Asn Lys Thr Ala 416
Thr Gly He Leu Tyr Asn Ala Thr Glu Gin Ser Tyr Lys Asp Gly Leu 432
Ser Lys Asn Asp Thr Arg 438
(7) INFORMATION FOR SEQ ID NO: 6
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 411
(B) TYPE: AMINO ACID
(ii) MOLECULE TYPE:
(A) DESCRIPTION: PROTEIN
(iii) HYPOTHETICAL: NO
(Vi) ORIGINAL SOURCE:
(A) ORGANISM: GUAR PLANT CYAMOPSIS TETRAGONOLOBA
(B) INDIVIDUAL ISOLATE: ALPHA-GALACTOSIDASE
(Xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6 Met Ala Thr His Tyr Ser He He Gly Gly Met He He Val Val Leu 16
Leu Met He He Gly Ser Glu Gly Gly Arg Leu Leu Glu Lys Lys Asn 32
Arg Thr Ser Ala Glu Ala Glu His Tyr Asn Val Arg Arg Tyr Leu Ala 48
Glu Asn Gly Leu Gly Gin Thr Pro Pro Met Gly Trp Asn Ser Trp Asn 64
His Phe Gly Cys Asp He Asn Glu Asn Val Val Arg Glu Thr Ala Asp 80
Ala Met Val Ser Thr Gly Leu Ala Ala Leu Gly Tyr Gin Tyr He Asn 96
Leu Asp Asp Cys Trp Ala Glu Leu Asn Arg Asp Ser Glu Gly Asn Met 112
Val Pro Asn Ala Ala Ala Phe Pro Ser Gly He Lys Ala Leu Ala Asp 128
Tyr Val His Ser Lys Gly Leu Lys Leu Gly Val Tyr Ser Asp Ala Gly 144
Asn Gin Thr Cys Ser Lys Arg Met Pro Gly Ser Leu Gly His Glu Glu 160
Gin Asp Ala Lys Thr Phe Ala Ser Trp Gly Val Asp Tyr Leu Lys Tyr 176
Asp Asn Cys Glu Asn Leu Gly He Ser Val Lys Glu Arg Tyr Pro Pro 192
Met Gly Lys Ala Leu Leu Ser Ser Gly Arg Pro He Phe Phe Ser Met 208
Cys Glu Trp Gly Trp Glu Asp Pro Gin He Trp Ala Lys Ser He Gly 224
Asn Ser Trp Arg Thr Thr Gly Asp He Glu Asp Asn Trp Asn Ser Met 240
Thr Ser He Ala Asp Ser Asn Asp Lys Trp Ala Ser Tyr Ala Gly Pro 256
Gly Gly Trp Asn Asp Pro Asp Met Leu Glu Val Gly Asn Gly Gly Met 272
Thr Thr Glu Glu Tyr Arg Ser His Phe Ser He Trp Ala Leu Ala Lys 288
Ala Pro Leu Leu Val Gly Cys Asp He Arg Ala Met Asp Asp Thr Thr 304
His Glu Leu He Ser Asn Ala Glu He Val Ala Val Asn Gin Asp Lys 320
Leu Gly Val Gin Gly Lys Lys Val Lys Ser Thr Aεn Asp Leu Glu Val 336
Trp Ala Gly Pro Leu Ser Asp Asn Lys Val Ala Val He Leu Trp Asn 352
Arg Ser Ser Ser Arg Ala Thr Val Thr Ala Ser Trp Ser Asp He Gly 368
Leu Gin Gin Gly Thr Thr Val Asp Ala Arg Asp Leu Trp Glu His Ser 384
Thr Gin Ser Leu Val Ser Gly Glu He Ser Ala Glu He Asp Ser His 400
Ala Cys Lys Met Tyr Val Leu Thr Pro Arg Ser 411 (8) INFORMATION FOR SEQ ID NO: 7
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 447
(B) TYPE: AMINO ACID
(ii) MOLECULE TYPE:
(A) DESCRIPTION: PROTEIN
(iii) HYPOTHETICAL: NO
(vi) ORIGINAL SOURCE:
(A) ORGANISM: ASPERGILLIS NIGER
(B) INDIVIDUAL ISOLATE: ALPHA-GALACTOSIDASE
(Xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7
Met He Gin Gly Leu Glu Ser He Met Asn Gin Gly Thr Lys Arg He 16
Leu Leu Ala Ala Thr Leu Ala Ala Thr Pro Trp Gin Val Tyr Gly Ser 32
He Glu Gin Pro Ser Leu Leu Pro Thr Pro Pro Met Gly Pro Asn Asn 48
Trp Ala Arg Phe Met Cys Asp Leu Asn Glu Thr Leu Phe Thr Glu Thr 64
Ala Asp Thr Met Ala Ala Asn Gly Leu Arg Asp Ala Gly Tyr Asn Arg 80
He Asn Leu Asp Asp Cys Trp Met Ala Tyr Gin Arg Ser Asp Asn Gly 96
Ser Leu Gin Trp Asn Thr Thr Lys Phe Pro His Gly Leu Pro Trp Leu 112
Ala Lys Tyr Val Lys Ala Lys Gly Phe His Phe Gly He Tyr Glu Asp 128
Ser Gly Asn Met Thr Cys Gly Gly Tyr Pro Gly Ser Tyr Asn His Glu 144
Glu Gin Asp Ala Asn Thr Phe Ala Ser Trp Gly He Asp Tyr Leu Lys 160
Leu Asp Gly Cys Asn Val Tyr Ala Thr Gin Gly Arg Thr Leu Glu Glu 176
Glu Tyr Lys Gin Arg Tyr Gly His Trp His Gin Val Leu Ser Lys Met 192
Gin His Pro Leu He Phe Ser Glu Ser Ala Pro Ala Tyr Phe Ala Gly 208
Thr Asp Asn Asn Thr Asp Trp Tyr Thr Val Met Asp Trp Val Pro He 224
Tyr Gly Glu Leu Ala Arg His Ser Thr Asp He Leu Val Tyr Ser Gly 240
Ala Gly Ser Ala Trp Asp Ser He Met Asn Asn Tyr Asn Tyr Asn Thr 256
Leu Leu Ala Arg Tyr Gin Arg Pro Gly Tyr Phe Asn Asp Pro Asp Phe 272
Leu He Pro Asp His Pro Gly Leu Thr Ala Asp Glu Lys Arg Ser His 288
Phe Ala Leu Trp Ala Ser Phe Ser Ala Pro Leu He He Ser Ala Tyr 304
He Pro Ala Leu Ser Lys Asp Glu He Ala Phe Leu He Asn Glu Ala 320 Leu He Ala Val Asn Gin Asp Pro Leu Ala Gin Gin Ala Thr Leu Ala 336
Ser Arg Asp Asp Thr Leu Asp He Leu Thr Arg Ser Leu Ala Asn Gly 352
Asp Arg Leu Leu Thr Val Leu Asn Lys Gly Asn Thr Thr Val Thr Arg 368
Asp He Pro Val Gin Trp Leu Gly Leu Thr Glu Thr Asp Cys Thr Tyr 384
Thr Ala Glu Asp Leu Trp Asp Gly Lys Thr Gin Lys He Ser Asp His 400
He Lys He Glu Leu Ala Ser His Ala Thr Ala Val Phe Arg Leu Ser 416
Leu Pro Gin Gly Cys Ser Ser Val Val Pro Thr Gly Leu Val Phe Asn 432
Thr Ala Ser Gly Asn Cys Leu Thr Ala Ala Ser Asn Ser Ser Val 447

Claims

WE CLAIM :
1. A recombinant chicken liver α-N-acetylgalacto¬ saminidase enzyme produced by Pichia pastoris.
2. A method of removing A antigens from the surface of erythrocytes comprising contacting said erythrocytes with a substantially purified, recombinant chicken liver α-N-acetyl¬ galactosaminidase enzyme produced by Pichia pastoris for a period of time sufficient to remove said A antigens from the surface of said erythrocytes.
3. A Pichia pastoris expression vector comprising a nucleic acid encoding chicken liver α-N-acetylgalactosaminidase enzyme.
4. A Pichia pastoris cell transformed with a vector comprising a nucleic acid encoding chicken liver α-N-acetyl¬ galactosaminidase enzyme.
5. A method for producing recombinant chicken liver α-N-acetylgalactosaminidase enzyme comprising culturing Pichia pastoris transformed with a vector comprising a nucleic acid encoding chicken liver α-N-acetylgalactosaminidase enzyme, and recovering α-N-acetylgalactosaminidase enzyme from the culture.
6. The recombinant α-N-acetylgalactosaminidase enzyme produced by the method of Claim 5.
7. The method of claim 6 which further comprises the step of purifying said α-N-acetylgalactosaminidase enzyme recovered from the culture using an affinity column.
8. The method of Claim 7, wherein said affinity column comprises aminocaproylgalactosylamine agarose.
PCT/US1996/017466 1995-10-18 1996-10-17 RECOMBINANT α-N-ACETYLGALACTOSAMINIDASE ENZYME WO1997014786A1 (en)

Priority Applications (1)

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AU77196/96A AU7719696A (en) 1995-10-18 1996-10-17 Recombinant alpha-n-acetylgalactosaminidase enzyme

Applications Claiming Priority (2)

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US54476995A 1995-10-18 1995-10-18
US08/544,769 1995-10-18

Publications (1)

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Cited By (1)

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US6135069A (en) * 1998-09-11 2000-10-24 Caterpillar Inc. Method for operation of a free piston engine

Citations (2)

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US4882279A (en) * 1985-10-25 1989-11-21 Phillips Petroleum Company Site selective genomic modification of yeast of the genus pichia
WO1994023070A1 (en) * 1993-03-26 1994-10-13 New York Blood Center, Inc. RECOMBINANT α-N-ACETYLGALACTOSAMINIDASE ENZYME AND cDNA ENCODING SAID ENZYME

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US4882279A (en) * 1985-10-25 1989-11-21 Phillips Petroleum Company Site selective genomic modification of yeast of the genus pichia
WO1994023070A1 (en) * 1993-03-26 1994-10-13 New York Blood Center, Inc. RECOMBINANT α-N-ACETYLGALACTOSAMINIDASE ENZYME AND cDNA ENCODING SAID ENZYME

Non-Patent Citations (4)

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Title
DAVIS et al., "Cloning and Sequencing of a Chicken Alpha-N-Acetylgalactosaminidase Gene", BIOCHIMICA BIOPHYSICS ACTA, November 1993, Vol. 1216, pages 296-298. *
ZHU A., "Trp-16 is Essential For the Activity of Alpha-Galactosidase and Alpha-N-Acetylgalactosaminidase", BIOCHIMICA BIOPHYSICA ACTA, September 1996, Vol. 1297, pages 99-104. *
ZHU et al., "Cloning and Characterization of a cDNA Encoding Chicken Liver Alpha-N-Acetylgalactosaminidase", December 1993, Vol. 137, pages 309-314. *
ZHU et al., "High-Level Expression and Purification of Coffe-Bean Alpha-Galactosidase Produced in the Yeast Pichia Pastoris", 01 December 1995, ARCH. BIOCHEM. BIOPHYS. Vol. 324, No. 1, pages 65-70. *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6135069A (en) * 1998-09-11 2000-10-24 Caterpillar Inc. Method for operation of a free piston engine

Also Published As

Publication number Publication date
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