WO1997014786A1 - α-N-ACETYLGALACTOSAMINIDASE RECOMBINEE - Google Patents

α-N-ACETYLGALACTOSAMINIDASE RECOMBINEE 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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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
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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

Cette invention concerne une enzyme recombinée destinée à être utilisée dans l'élimination d'antigènes A de la surface de cellules dans des produits du sang. Spécifiquement, cette invention concerne une α-N-acétylgalactosaminidase recombinée de foie de poulet, des procédés de clônage et d'expression de ladite α-N-acétylgalactosaminidase recombinée ainsi qu'un procédé d'élimination d'antigènes A de la surface de cellules dans des produits du sang, à l'aide de ladite α-N-acétylgalactosaminidase recombinée.
PCT/US1996/017466 1995-10-18 1996-10-17 α-N-ACETYLGALACTOSAMINIDASE RECOMBINEE WO1997014786A1 (fr)

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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

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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 (fr) * 1993-03-26 1994-10-13 New York Blood Center, Inc. α-N-ACETYLGALACTOSAMINIDASE RECOMBINEE ET ADNc CODANT CETTE ENZYME

Patent Citations (2)

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