WO1996023869A1 - ENZYME α-GALACTOSIDASE RECOMBINEE - Google Patents

ENZYME α-GALACTOSIDASE RECOMBINEE Download PDF

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Publication number
WO1996023869A1
WO1996023869A1 PCT/US1996/001212 US9601212W WO9623869A1 WO 1996023869 A1 WO1996023869 A1 WO 1996023869A1 US 9601212 W US9601212 W US 9601212W WO 9623869 A1 WO9623869 A1 WO 9623869A1
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Prior art keywords
galactosidase
leu
gly
ala
ser
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PCT/US1996/001212
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English (en)
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Alex Zhu
Jack Goldstein
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New York Blood Center, Inc.
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Priority to EP96903741A priority Critical patent/EP0807165A4/fr
Priority to JP8523663A priority patent/JPH10513057A/ja
Priority to AU47725/96A priority patent/AU4772596A/en
Publication of WO1996023869A1 publication Critical patent/WO1996023869A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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)
    • C12N9/2465—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1) acting on alpha-galactose-glycoside bonds, e.g. alpha-galactosidase (3.2.1.22)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/14—Blood; Artificial blood
    • A61K35/18—Erythrocytes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/80—Vectors or expression systems specially adapted for eukaryotic hosts for fungi
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • This invention relates to a recombinant enzyme for use in the removal of type B antigens from the surface of cells in blood products, thereby converting type B blood products to type O blood products and type AB blood products to type A blood products without otherwise affecting the structure and function of the cells in the blood products.
  • This invention further relates to methods of cloning and expressing said recombinant enzyme.
  • this invention is directed to a recombinant coffee bean ⁇ -galactosidase enzyme, a recombinant vector which encodes coffee bean ⁇ -galactosidase, methods of cloning and expressing said recombinant ⁇ -galactosidase enzyme, the use of said recombinant ⁇ -galactosidase enzyme to cleave galactose sugar residues, most particularly ⁇ l,3-linked galactose residues, which are responsible for blood group B specificity, and a method of removing type B antigens from the surface of cells in type B and AB blood products using said recombinant coffee bean ⁇ -galactosidase enzyme by contacting said enzyme with blood products so as to remove the terminal moiety of the B-antigenic determinant from the surface of cells (for example, erythrocytes) in said blood products.
  • the recombinant coffee bean ⁇ -galactosidase enzyme of this invention provides a readily available and cost-efficient enzyme which can be used in the removal of type B antigens from the surface of cells in type B and AB blood products.
  • Treatment of type B blood products with the recombinant enzyme of this invention provides a source of cells free of the B antigen, which blood products are thereby rendered useful in transfusion therapy in the same manner as 0 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 0 contains neither antigen, but does contain a structure known as H 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.
  • 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.
  • 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 O 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 O.
  • type O blood is considered "universal", and may be used for all transfusions.
  • the process for converting B and AB erythrocytes which is described in the '619 Patent includes the steps of equilibrating B or AB erythrocytes, contacting the equilibrated erythrocytes and purified ⁇ -galactosidase for a period of time sufficient to convert the B antigen in the erythrocytes to the H antigen, removing the ⁇ -galactosidase enzyme from the erythrocytes and re-equilibrating the erythrocytes.
  • ⁇ -galactosidase enzymes from a number of sources have been purified, sequenced, cloned and expressed.
  • 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.
  • 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 B antigens from the surface of cells, the enzyme must be sufficiently active at or above a pH at which the cells being treated that can be maintained, that being pH 5.6 (or above) for red cells. Therefore, the pH optimum and activity profile of an appropriate enzyme must still provide reasonable enzyme activity at this pH.
  • the pH optimum of Ehrlich cell ⁇ -galactosidase enzyme centers near 4.5, irrespective of substrate (see Yagi et al., Archives Biochem. and Biophysics. Vol. 280, pp. 61-67 (1990)).
  • the pH optimum or Ehrlich cell ⁇ -galactosidase has been found to be 4.5 for water-soluble fluorogenic substrates and oligosaccharides (see Dean et al., J. Biol. Chem.. Vol. 254, pp. 10006-10010 (1979)).
  • Coffee bean ⁇ -galactosidase enzyme shows a Vmax/km value of 236 at pH 6.0 toward PNP- ⁇ -gal, whereas ⁇ -galactosidases isolated from human cells (see Dean and Sweeley, J. Biol. Chem.. Vol. 254, pp. 10006-10010 (1979)) or Ehrlich ascites tumor (see Yagi et al., Archives Biochem. and Biophysics. Vol. 280, pp.
  • coffee bean ⁇ -galactosidase showed a relatively broad substrate specificity, suggesting that it is suited for cleaving many kinds of terminal ⁇ -galactosyl linkages.
  • the ⁇ -galactosidases studied the one obtained from coffee bean demonstrates the highest activity in removing terminal ⁇ l,3-linked galactose residues from glycoconjugates. This makes the coffee bean enzyme a most appropriate enzyme in the study and performance of enzymatic blood conversion.
  • a recombinant, cloned enzyme would allow for specific protein sequence modifications, which can be introduced in order to generate an enzyme with further optimized specific activity, substrate specificity and pH range. It is therefore an object of this invention to provide recombinant coffee bean ⁇ -galactosidase enzyme for use in the removal of B antigens from the surface of cells in blood products.
  • This invention is directed to a recombinant coffee bean ⁇ -galactosidase enzyme capable of cleaving ⁇ l,3-linked glycoside linkages on cells.
  • This invention is further directed to a recombinant vector containing a nucleotide sequence encoding coffee bean ⁇ -galactosidase.
  • this invention is directed to a method of producing coffee bean ⁇ -galactosidase, and to a method of removing B antigens from the surface of cells which method comprises contacting cells with recombinant coffee bean ⁇ -galactosidase enzyme for a period of time sufficient to remove the B antigens from the surface of the cells.
  • Figure l represents the nucleotide and deduced amino acid sequence of full-length cDNA encoding coffee bean ⁇ -galactosidase
  • Figure 2 represents a comparison of sequence homology of ⁇ -galactosidase from coffee bean, guar iCvamopsis tetraqonoloba) , human placenta, yeast (Saccharomvces cerevisiae) and fungi (Asperqillus ni ⁇ eri as aligned using the computer program PROSIS and manual arrangement;
  • Figure 3 represents immunoprecipitation with polyclonal antibody of cloned coffee bean ⁇ -galactosidase expressed jLn vitro in rabbit reticulocyte lysate and wheat germ extract, as analyzed by SDS-PAGE and autoradiographed;
  • Figure 4 represents Western blot analysis of recombinant coffee bean ⁇ -galactosidase expressed in transfected sf9 insect cells using antibody against purified coffee bean ⁇ -galactosidase.
  • Figure 5 represents the sequence of the plasmid p ⁇ F-BZ around the 5' cloning site (EcoRI) of the insert. The two arrows indicate the signal cleavage sites as indicated by N-terminal sequencing of the secreted enzyme.
  • Figure 6 represents the chromatogram from the cation exchange chromatography purification of the recombinant ⁇ -galactosidase enzyme produced by the Pichia pastoris expression system.
  • Figure 7 represents the SDS-PAGE analysis of the chromatography purified recombinant ⁇ -galactosidase enzyme produced by the Pichia pastoris expression system.
  • Lane l supernatant of Pichia pastoris culture; lane 2, unbound fraction from the column; lane 3, fraction #55; lane 4, fraction #65; lane 5, fraction #80; lane 6, fraction #150; lane 7, native ⁇ -galactosidase enzyme; and lane 8, size markers (kDa) .
  • This invention is directed to a recombinant coffee bean ⁇ -galactosidase enzyme capable of cleaving ⁇ l,3-linked glycoside linkages on cells.
  • the recombinant coffee bean ⁇ -galactosidase enzyme of the invention has a molecular weight of about 42 kDa, and has about 80% amino acid sequence homology with guar ⁇ -galactosidase enzyme.
  • This invention is further directed to a recombinant vector containing a nucleotide sequence which encodes coffee bean ⁇ -galactosidase.
  • this invention is directed to a method of producing coffee bean ⁇ -galactosidase, and to a method of removing B antigens from the surface of cells which method comprises contacting cells with a recombinant coffee bean ⁇ -galactosidase enzyme for a period of time sufficient to remove the B antigens from the surface of the cells.
  • Group B erythrocytes may be treated with ⁇ -galactosidase isolated from coffee beans to cleave the terminal ⁇ l,3-linked galactose residues responsible for blood group B specificity in order to convert the group B erythrocytes serologically to group O erythrocytes.
  • ⁇ -galactosidase isolated from coffee beans to cleave the terminal ⁇ l,3-linked galactose residues responsible for blood group B specificity in order to convert the group B erythrocytes serologically to group O erythrocytes.
  • the inventors In order to produce purified ⁇ -galactosidase enzyme in large quantities and improve its enzymatic properties, the inventors have isolated the cDNA clone for coffee bean ⁇ -galactosidase.
  • ⁇ -galactosidase has been purified from several sources. However, only coffee bean ⁇ -galactosidase cleaves ⁇ l,3-linked galactose residues responsible for blood group B specificity. Hence, only coffee bean ⁇ -galactosidase can be used to convert type B blood products to type 0 blood products, and type AB blood products to type A blood products.
  • the full length cDNA which encodes coffee bean ⁇ -galactosidase is represented in SEQ ID N0:l and Figure 1.
  • a DNA vector containing a sequence encoding coffee bean ⁇ -galactosidase was deposited under the Budapest Treaty with the American Type Culture Collection, Rockville, Maryland, on September 8, 1993, tested and found viable on September 14, 1993, and catalogued as ATCC #75556.
  • expression vectors containing the coffee bean ⁇ -galactosidase coding sequence can be used to construct expression vectors containing the coffee bean ⁇ -galactosidase 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 coffee bean ⁇ -galactosidase 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 coffee bean ⁇ -galactosidase 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.
  • a recombinant coffee bean ⁇ -galactosidase enzyme is cloned and expressed, said enzyme can be used to remove B antigens from the surface of cells in blood products.
  • Type B antigens can be removed from the surface of erythrocytes by contacting the erythrocytes with the recombinant coffee bean ⁇ -galactosidase enzyme of the invention for a period of time sufficient to remove the B antigens from the surface of the erythrocytes.
  • Example 1 In order to assess the relative abilities of ⁇ -galactosidase enzyme isolated from different sources to remove ⁇ l,3-linked galactose residues from red cells, 100 ml of type B red blood cells were treated with isolated ⁇ -galactosidases from yeast (S. cerevisiae) , fungi (A. niger) , guar (C . tetraqonoloba) and coffee bean. The treatment conditions and digestion results are provided below in Table I, below. Digestion of terminal sugars ( ⁇ l,3-linked galactose residues) was determined by assessing reduction or elimination of the agglutination of treated cells in the presence of polyclonal anti-B antibody. No detectable change in agglutination indicated no digestion. TABLE I
  • coffee bean ⁇ -galactosidase was purified to apparent homogeneity from green coffee beans.
  • the procedure used for purification of ⁇ -galactosidase from coffee beans was developed and optimized in the laboratory to provide pure ⁇ -galactosidase enzyme (demonstrating a single band on
  • PCS buffer pH 5.6 had the following composition: 58 mM dibasic sodium phosphate, 21 mM citric acid, 77 mM sodium chloride.
  • Concentrate phosphate-citrate buffer was prepared by titrating 50 mM citric acid with 100 mM dibasic sodium phosphate pH 3.7.
  • Sepharose divinylsulfone galactose was prepared according to the procedure described by Ersson et al., Biochem. et Biophys. Acta. Vol. 494, pp. 51-60 (1977) , which is incorporated herein by reference.
  • Polybuffer exchanger 4 is available from Pharmacia, Inc.
  • Other similar anion exchange resins which are well known in the art and available commercially, can be used in place of DE53 in the above procedure, particularly other DEAE (diethylaminoethyl) resins.
  • Alternative methods for purification of ⁇ -galactosidase from coffee beans have been reported by Haibach et al., Biochem. et Biophvs. Res. Comm.. Vol. 181, No. 3, pp. 1564-1571 (1991) and Harpaz et al., Biochem et Biophvs. Acta. Vol. 341, pp.
  • Microsequencing of the unblocked mature enzyme provided an amino-terminal sequence (N-pep) of 19 residues.
  • N-pep amino-terminal sequence
  • 0.2 mg of the purified ⁇ -galactosidase was treated with 2 mg of cyanogen bromide in 70% formic acid for 24 hours at room temperature in the dark.
  • the peptides were isolated by reverse phase HPLC.
  • Two peptide sequences, 2-pep and 3-pep, were then determined by automated gas phase microsequencing.
  • the sequences of the three peptides are indicated in Figure 1.
  • Figure 1 represents the full length cDNA encoding coffee bean ⁇ -galactosidase.
  • the first 15 amino acids comprise a putative signal peptide which is cleaved during biosynthesis. Therefore, the mature coffee bean ⁇ -galactosidase enzyme is comprised of the amino acids 16-378 of Figure 1.
  • the potential N-linked glycosylation site is double-underlined at amino acid residues 160-162.
  • the polyadenylation signal (AATAAA) at the position ntl361-1366 is boxed.
  • the oligonucleotides, CB1, CB4 through CB9, are shown with arrows to indicate 5' to 3' direction.
  • the CB1* was designed as 5 , ACA(CT)CCA(T)CCA(T)ATGGNTGGAA.
  • the CB4* based on the sequence of the peptide, 3-pep, has the sequence 5 , -TGT(A)GGT(GA)GTNAGG(CA)ACG(A)TACAT.
  • CB1 bears the least codon degeneracies in the peptide sequence of N-pep as determined by the computer program "Primer” (Scientific and Educational Software, Inc.).
  • RNA was prepared from 2 grams of dried green coffee beans by using the Extract-A-plant RNA Isolation kit (ClonTech) according to the manufacturer's procedure. The quality of the isolated RNA was confirmed by denaturing agarose gel electrophoresis. The messenger RNA was purified from the total RNA by using an oligo-dT column (ClonTech) .
  • cDNA using isolated coffee bean mRNA was prepared according to the standard procedure known in the art for reverse transcription.
  • a mixture of oligo dT and random primer was used as the reverse transcription primer in the reaction to avoid the 3' bias when oligo-dT is used alone.
  • the cDNA then provided the template in a PCR reaction for 35 cycles, 94°C 1 minute, 50°C 2 minutes and 72°C 3 minutes. In the presence of oligonucleotides CB1 and CB4, this PCR procedure produced a fragment of approximately l.lkb.
  • BZ This fragment, designated BZ, was cloned directly into the pCRII vector (Invitrogen) for further analysis.
  • its deduced amino acid sequence matched the peptide sequences obtained from purified coffee bean ⁇ -galactosidase, providing evidence for its authenticity as coffee bean ⁇ -galactosidase cDNA.
  • the second oligonucleotide, CB6, together with the universal primer was used to amplify the 5' region upstream of the coding sequence by PCR. Since no distinctive DNA band was visible on the agarose gel after two PCR amplifications, the PCR product mixture was cloned into the pCRII vector and screened by hybridizing the colonies with the radioactively labeled l.lkb fragment BZ (sequence ntl68-1234 in Figure 1) . The positive colonies were picked for plasmid preparation.
  • the sequencing of the plasmid indicated that the DNA fragment, designated 5'BZ, obtained by the 5' RACE technique contained a 240bp overlap (the sequence between CB1 and CB6) with the BZ, and about a 170bp further upstream sequence which includes the N-te ⁇ ninus of the mature enzyme and the putative signal peptide sequence.
  • the cDNA was reverse-transcribed from coffee bean mRNA by using a primer, PI, which has the sequence 5'-GACTCGAGTCGACATCGA-(T) 17 .
  • PCR amplification was then carried out with a specific primer CB7 (sequence nt940-957 in Figure 1) and an adapter primer, PII.
  • PII has the same sequence as PI except that it lacks seventeen thymidine residues at its 3' end.
  • the PCR product was analyzed on 1% low melting point agarose gel and a distinctive band of about 500 bp long was visualized.
  • the fragment designated 3' BZ was isolated and cloned into the pCRII vector for sequencing.
  • sequence data indicated that the 5' region of the fragment, 3 , BZ, is identical to the 3' region (sequence from nt940-1234) of the BZ.
  • An in-frame stop codon TGA was localized at ntl236-1238, confirming that the peptide sequence, 3-pep, obtained from the purified coffee bean ⁇ -galactosidase represents the C-terminal sequence of the protein.
  • the three DNA fragments, 5'BZ, BZ and 3'BZ, were linked together by PCR to reconstitute the full length clone for coffee bean ⁇ -galactosidase.
  • Oligonucleotide CB9 was made, which corresponded to sequence nt77-94 shown in Figure 1.
  • cDNA was synthesized from coffee bean mRNA using the 3' RACE technique as previously described.
  • a l.35kb fragment was amplified by PCR using two primers CB9 and PII, and was then cloned into the pCR II vector.
  • the plasmids thus generated contained the 1.35kb insert in both orientations.
  • the plasmid pCR-BZ6 has the insert downstream of an SP6 promoter, which was used in in vitro expression.
  • a second plasmid pCR-BZ7 containing the opposite insert orientation was used for subcloning the ⁇ -galactosidase cDNA into a baculovirus expression vector.
  • the sequence of the 1.35kb product matched with the corresponding sequences from the three separate fragments, 5'BZ, BZ and 3'BZ, confirming the authentic sequence of the coffee bean ⁇ -galactosidase cDNA shown in Figure 1.
  • the coffee bean ⁇ -galactosidase cDNA clone was characterized.
  • the sequence shown in the Figure 1 encodes a protein having a molecular weight of 42kDa, which closely approximates the size of the purified coffee bean ⁇ -galactosidase as estimated on SDS-PAGE.
  • Three peptide sequences, N-pep, 2-pep and 3-pep, which were derived from purified enzyme, are underlined in Figure l. These sequences matched the deduced amino acid sequences. This -19- confirms that the cDNA clone isolated from coffee bean RNA encodes ⁇ -galactosidase.
  • the first plant ⁇ -galactosidase cDNA was cloned from guar (see Overbeeke et al., Plant Molecular Biology. Vol. 13, pp. 541-550 (1989)). Guar ⁇ -galactosidase encodes a protein of 411 residues having a molecular weight of 45 kDa. Although both ⁇ -galactosidases from guar and coffee bean show comparable activities toward the synthetic substrate PNP- ⁇ -gal, their specificities toward oligosaccharide chains are very different.
  • Guar ⁇ -galactosidase primarily cleaves a 1,6 glycoside linkages, whereas coffee bean ⁇ -galactosidase cleaves ⁇ 1,3 and 1,4 linkages. Thus, the guar ⁇ -galactosidase is unable to cleave significant amounts of terminal ⁇ l,3-linked galactose residues from the cell surface of B group red cells.
  • Figure 2 represents the sequence homology of ⁇ -galactosidase from different sources.
  • amino acid sequences of ⁇ -galactosidase from coffee bean (coffee) , Cvamopsis tetragonoloba (guar) , human placenta (human) , Saccharomvces cerevisiae (yeast) and Aspergillus niger (Aspergillus) were aligned by using the computer program PROSIS (Hitachi Software Engineering Corp., Ltd.) and manual arrangement. The gaps are created in order to show maximum similarity. The numbers above the sequences indicate the relative position of each amino acid sequence.
  • yeast and Aspergillus niger ⁇ -galactosidases are truncated at the C-terminus, (indicated by *) , removing 38 and 103 residues respectively.
  • Identical or conservatively substituted amino acid residues are boxed according to the equivalent amino acid list. 1: A,S,T,P and G; 2: N,D,E and Q; 3: H,R and K; 4: M,L,I and V; and 5: F,Y and W.
  • Figure 3 represents in vitro expression and immunoprecipitation of cloned coffee bean ⁇ -galactosidase.
  • plasmids pCR and pCR-BZ6
  • TNT rabbit reticulocyte lysate in the presence of 35 S-methionine and SP6 DNA polymerase according to the Promega recommended protocol.
  • the samples 5 ml of each reaction were loaded onto a 12% gel SDS-PAGE (lanes 2 and 3) .
  • Immunoprecipitation was carried out by incubating the same expression mixtures (20 ml) with antisera (1 ml) raised against purified coffee bean ⁇ -galactosidase. The immunoprecipitated samples were analyzed in lanes 4 and 5. Lanes 6 through 9 show the results of the same experiments except that TNT wheat germ extract was used instead of rabbit reticulocyte lysate. The arrow at right indicates the expressed ⁇ -galactosidase. The molecular weight standard (lane 1) is shown at left.
  • Coffee bean ⁇ -galactosidase was then functionally expressed in insect cells. Many eukaryotic proteins have been expressed in insect cells infected with recombinant baculovirus (see King et al., The Baculovirus Expression
  • Coffee bean ⁇ -galactosidase cDNA was subcloned from the plasmid pCR-BZ7 into the unique Notl/BamHI sites of a baculovirus expression vector pVL 1392 (PharMingen) , generating the plasmid pVL-BZ. Expression of ⁇ -galactosidase cDNA was thus under the control of a strong viral promoter (polyhedrin promoter) .
  • the plasmid pVL-BZ was co-transfected into sf9 insect cells with baculoGold DNA (PharMingen) , a lethal deletion of the virus DNA, according to the procedure suggested by the manufacturer.
  • viable virus containing the ⁇ -galactosidase cDNA was thus reconstituted inside the insect cells and released into the medium.
  • the transfection supernatant (1 ml) was then added to fresh sf9 cells (2 x 10 6 ) . After incubation at 27°C for three days, the supernatant was harvested and used for virus amplification one more time in order to obtain a high titer of virus.
  • FIG. 4 represents expression of recombinant coffee bean ⁇ -galactosidase in insect cells (sf9) .
  • the supernatants and cells were collected after the second amplification and analyzed by Western blot with polyclonal antibody against purified coffee bean ⁇ -galactosidase.
  • the supernatant and cells from pVL-BZ transfection are shown in lanes 1 and 2, respectively.
  • the supernatant (lane 3) and cells (lane 4) from wild-type virus transfection were used as a negative control.
  • Lane 5 is ⁇ -galactosidase purified from coffee bean. Molecular weight standards are listed at left.
  • the coffee bean ⁇ -galactosidase was expressed in the sf9 cells transfected with the plasmid pVL-BZ (lane 2) but not in cells transfected with wild-type virus (lane 4) . Its migration on the gel was similar to that of purified enzyme (lane 5) . In addition, secretion of the expressed protein in the culture supernatant (lane 1) was not detected.
  • the ⁇ -galactosidase activity was tested by directly incubating pVL-BZ transfected sf9 cells with 1.25 mM PNP-a-gal (pH 6.5). During the incubation, the PNP- ⁇ -galactosidase substrate enters the cells by diffusion. After incubation at 37°C for an hour the reaction was stopped by adding 1 ml of borate buffer (pH 9.8) and absorbance at 405 n was measured. The total proteins in the reaction were precipitated by addition of trichloroacetate and measured by Bio-Rad Protein Assay (Bio-Rad) .
  • the average activity of ⁇ -galactosidase expressed in the insect cells was approximately 300 units, one unit being defined as 1 nmol of substrate hydrolyzed per hour at 37°C.
  • the endogenous activity in the wild-type virus infected cells was undetectable under such conditions.
  • the recombinant coffee bean ⁇ -galactosidase enzyme of the invention can be used to remove terminal galactose residues from the non-reducing end of carbohydrate chains (from polysaccharides and glycoconjugates) , particularly those galactose residues which are ⁇ l,3-linked.
  • the recombinant coffee bean ⁇ -galactosidase enzyme of the invention can be used to convert type B erythrocytes to type O and type AB erythrocytes to type A. Specifically, the recombinant coffee bean ⁇ -galactosidase enzyme of the invention is put into contact with cells having B antigenicity for a period of time sufficient to remove the B antigens from the surface of the cells.
  • erythrocytes having B antigenicity are washed and then equilibrated in isotonic phosphate-citrate-sodium chloride (PCS) at pH 5.5 and 5.6, sequentially, ⁇ -galactosidase is added at a concentration of from 75 to 200 U/ml, and the mixture is incubated at 26°C. More preferably, the ⁇ -galactosidase is added at a concentration of 200 U/ml and the mixture is incubated at 26°C for 2.25 hours. Most preferably, the final hematocrit of the erythrocytes is 65 to 75%.
  • PCS isotonic phosphate-citrate-sodium chloride
  • cells transformed with a recombinant vector which encodes coffee bean ⁇ -galactosidase can be cultured and coffee bean ⁇ -galactosidase can be recovered from the culture, which coffee bean ⁇ -galactosidase can then be used to remove B antigens from the surface of cells and blood products.
  • Coffee Bean ⁇ -Galactosidase in Pichia pastoris Coffee bean ⁇ -galactosidase cDNA was subcloned in the EcoRI site of Pichia pastoris expression vector pPIC9 (Invitrogen Corp. , San Diego, CA) generating the plasmid p ⁇ F-BZ with the sequence around the 5' cloning site of the insert as shown in Figure 5.
  • the expression of ⁇ - galactosidase was under the control of the methanol inducable promoter AOX1.
  • the expressed protein was secreted into the culture media via the signal sequence of yeast ⁇ mating factor.
  • N-terminal sequencing of the purified protein suggested that the signal sequence was cleaved at two positions as indicated by the two arrows in Figure 5, generating two forms of ⁇ -galactosidase with Phe or Leu as the N-terminal residue in approximately equal molarity.
  • Characterization of the native coffee bean ⁇ -galactosidase and the recombinant coffee bean ⁇ -galactosidase indicated that despite the mixture of N-terminal amino acid residues, the recombinant ⁇ -galactosidase exhibited essentially the same molecular size, specific activity, pH optima and kinetic parameters as the native enzyme.
  • ATC AAT CTT GAT GAC TGT TGG GCA GAA CTT AAC AGA GAT TCA CAG GGG 335 lie Asn Leu Asp Asp Cys Trp Ala Glu Leu Asn Arg Asp Ser Gin Gly 65 70 75
  • GCA AAA GCA CCT CTA CTG ATT GGC TGT GAC ATT CGA TCC ATG GAC GGT 911 Ala Lys Ala Pro Leu Leu lie Gly Cys Asp lie Arg Ser Met Asp Gly 245 250 255 260
  • Leu Met lie lie Gly Ser Glu Gly Gly Arg Leu Leu Glu Lys Lys Asn 20 25 30
  • Leu Lys Leu Gly lie Tyr Ala Asp Val Gly Asn Lys Thr Cys Ala Gly 130 135 140
  • Leu Leu Asp Thr Ala Asp Arg lie Ser Asp Leu Gly Leu Lys Asp Met 50 55 60
  • Gly Tyr Lys Tyr lie lie Leu Asp Asp Cys Trp Ser Ser Gly Arg Asp 65 70 75 80
  • Val Asp Asn Ser Thr Ala Ser Ala lie Leu Gly Arg Asn Lys Thr Ala

Abstract

Cette invention se rapporte à une enzyme recombinée utile dans l'enlèvement des antigènes de type B de la surface de cellules dans des produits sanguins. Cette invention concerne notamment une enzyme α-galactosidase recombinée du grain de café, un vecteur recombiné codant l'α-galactosidase du grain de café, des méthodes de clonage et d'expression de l'α-galactosidase recombinée du grain de café ainsi qu'un procédé d'enlèvement d'antigènes B de la surface de cellules dans des produits sanguins par utilisation de ladite α-galactosidase.
PCT/US1996/001212 1995-01-30 1996-01-30 ENZYME α-GALACTOSIDASE RECOMBINEE WO1996023869A1 (fr)

Priority Applications (3)

Application Number Priority Date Filing Date Title
EP96903741A EP0807165A4 (fr) 1995-01-30 1996-01-30 ENZYME -g(a)-GALACTOSIDASE RECOMBINEE
JP8523663A JPH10513057A (ja) 1995-01-30 1996-01-30 組換えα−ガラクトシダーゼ酵素
AU47725/96A AU4772596A (en) 1995-01-30 1996-01-30 Recombinant alpha-galactosidase enzyme

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US38019495A 1995-01-30 1995-01-30
US08/380,194 1995-01-30

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WO2003027245A3 (fr) * 2001-09-25 2003-07-10 Zymequest Inc CONVERSION ENZYMATIQUE DES GROUPES SANGUINS A ET B, ET ERYTHROCYTES AB UTILISANT α-N-ACETYLGALACTOSAMINIDASES ET α-GALACTOSIDASES AYANT DES SPECIFICITES DE SUBSTRAT ET DES PROPRIETES CINETIQUES UNIQUES
WO2011061736A1 (fr) * 2009-11-17 2011-05-26 Protalix Ltd. Alpha galactosidase alcaline pour le traitement de la maladie de fabry
US8742079B2 (en) 2007-08-20 2014-06-03 Protalix Ltd. Saccharide-containing protein conjugates and uses thereof
US9194011B2 (en) 2009-11-17 2015-11-24 Protalix Ltd. Stabilized alpha-galactosidase and uses thereof
US9732333B2 (en) 2011-01-20 2017-08-15 Protalix Ltd. Nucleic acid construct for expression of alpha-galactosidase in plants and plant cells
CN114752581A (zh) * 2022-04-20 2022-07-15 南京工业大学 一种α-半乳糖苷酶突变体及其应用

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US7767415B2 (en) 2001-09-25 2010-08-03 Velico Medical, Inc. Compositions and methods for modifying blood cell carbohydrates
WO2003027245A3 (fr) * 2001-09-25 2003-07-10 Zymequest Inc CONVERSION ENZYMATIQUE DES GROUPES SANGUINS A ET B, ET ERYTHROCYTES AB UTILISANT α-N-ACETYLGALACTOSAMINIDASES ET α-GALACTOSIDASES AYANT DES SPECIFICITES DE SUBSTRAT ET DES PROPRIETES CINETIQUES UNIQUES
US7993896B2 (en) 2001-09-25 2011-08-09 Velico Medical, Inc. Streptomyces griseoplanus α-galactosidases for removing immunodominant α-galactose monosaccharides from blood group B or AB reactive cells
US8697411B2 (en) 2001-09-25 2014-04-15 Velico Medical, Inc. Streptomyces griseoplanus comprising an α-galactosidase for removing immunodominant α-galactose monosaccharides from blood group B or AB reactive cells
US8742079B2 (en) 2007-08-20 2014-06-03 Protalix Ltd. Saccharide-containing protein conjugates and uses thereof
US9194011B2 (en) 2009-11-17 2015-11-24 Protalix Ltd. Stabilized alpha-galactosidase and uses thereof
WO2011061736A1 (fr) * 2009-11-17 2011-05-26 Protalix Ltd. Alpha galactosidase alcaline pour le traitement de la maladie de fabry
US9708595B2 (en) 2009-11-17 2017-07-18 Protalix Ltd. Stabilized alpha-galactosidase and uses thereof
US10280414B2 (en) 2009-11-17 2019-05-07 Protalix Ltd. Stabilized α-galactosidase and uses thereof
US10870842B2 (en) 2009-11-17 2020-12-22 Protalix Ltd. Stabilized alpha-galactosidase and uses thereof
US9732333B2 (en) 2011-01-20 2017-08-15 Protalix Ltd. Nucleic acid construct for expression of alpha-galactosidase in plants and plant cells
CN114752581A (zh) * 2022-04-20 2022-07-15 南京工业大学 一种α-半乳糖苷酶突变体及其应用
CN114752581B (zh) * 2022-04-20 2023-05-26 南京工业大学 一种α-半乳糖苷酶突变体及其应用

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EP0807165A1 (fr) 1997-11-19
EP0807165A4 (fr) 1998-07-15
AU4772596A (en) 1996-08-21
CA2211417A1 (fr) 1996-08-08
JPH10513057A (ja) 1998-12-15

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