EP0807165A1 - ENZYME $g(a)-GALACTOSIDASE RECOMBINEE - Google Patents

ENZYME $g(a)-GALACTOSIDASE RECOMBINEE

Info

Publication number
EP0807165A1
EP0807165A1 EP96903741A EP96903741A EP0807165A1 EP 0807165 A1 EP0807165 A1 EP 0807165A1 EP 96903741 A EP96903741 A EP 96903741A EP 96903741 A EP96903741 A EP 96903741A EP 0807165 A1 EP0807165 A1 EP 0807165A1
Authority
EP
European Patent Office
Prior art keywords
galactosidase
leu
gly
ala
ser
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP96903741A
Other languages
German (de)
English (en)
Other versions
EP0807165A4 (fr
Inventor
Alex Zhu
Jack Goldstein
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
New York Blood Center Inc
Original Assignee
New York Blood Center Inc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by New York Blood Center Inc filed Critical New York Blood Center Inc
Publication of EP0807165A1 publication Critical patent/EP0807165A1/fr
Publication of EP0807165A4 publication Critical patent/EP0807165A4/fr
Withdrawn legal-status Critical Current

Links

Classifications

    • 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 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.
  • 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.
  • 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.
  • 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 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;
  • 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.
  • 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
  • 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.
  • 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 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.
  • 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) .
  • 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.
  • 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.

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.
EP96903741A 1995-01-30 1996-01-30 ENZYME -g(a)-GALACTOSIDASE RECOMBINEE Withdrawn EP0807165A4 (fr)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
US380194 1982-05-20
US38019495A 1995-01-30 1995-01-30
PCT/US1996/001212 WO1996023869A1 (fr) 1995-01-30 1996-01-30 ENZYME α-GALACTOSIDASE RECOMBINEE

Publications (2)

Publication Number Publication Date
EP0807165A1 true EP0807165A1 (fr) 1997-11-19
EP0807165A4 EP0807165A4 (fr) 1998-07-15

Family

ID=23500271

Family Applications (1)

Application Number Title Priority Date Filing Date
EP96903741A Withdrawn EP0807165A4 (fr) 1995-01-30 1996-01-30 ENZYME -g(a)-GALACTOSIDASE RECOMBINEE

Country Status (5)

Country Link
EP (1) EP0807165A4 (fr)
JP (1) JPH10513057A (fr)
AU (1) AU4772596A (fr)
CA (1) CA2211417A1 (fr)
WO (1) WO1996023869A1 (fr)

Families Citing this family (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6458574B1 (en) * 1996-09-12 2002-10-01 Transkaryotic Therapies, Inc. Treatment of a α-galactosidase a deficiency
US7767415B2 (en) 2001-09-25 2010-08-03 Velico Medical, Inc. Compositions and methods for modifying blood cell carbohydrates
CA2696699A1 (fr) 2007-08-20 2009-02-26 Protalix Ltd. Conjugues de proteines contenant un saccharide et leurs utilisations
US20120230974A1 (en) * 2009-11-17 2012-09-13 Protalix Ltd Alkaline alpha galactosidase for the treatment of fabry disease
US9194011B2 (en) 2009-11-17 2015-11-24 Protalix Ltd. Stabilized alpha-galactosidase and uses thereof
CN103443270B (zh) 2011-01-20 2017-06-06 普罗塔里克斯有限公司 用于在植物和植物细胞中表达α‑半乳糖苷酶的核酸构建体
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

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1987007641A1 (fr) * 1986-06-03 1987-12-17 Unilever Nv Production de alpha-galactosidase de guar par des hotes transformes par des procedes d'adn recombinant
WO1994009123A1 (fr) * 1992-10-22 1994-04-28 The New York Blood Center, Inc. Recyclage d'enzymes utilisees dans la conversion d'erythrocytes des sous-types a, b et ab

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE69133066T2 (de) * 1990-01-16 2003-03-06 Ct Ingenieria Genetica Biotech Verfahren zur Expression heterologischer Gene in der Hefe Pichia Pastoris, Expressionsverfahren und transformierte Mikroorganismen
US5330901A (en) * 1991-04-26 1994-07-19 Research Corporation Technologies, Inc. Expression of human serum albumin in Pichia pastoris

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1987007641A1 (fr) * 1986-06-03 1987-12-17 Unilever Nv Production de alpha-galactosidase de guar par des hotes transformes par des procedes d'adn recombinant
WO1994009123A1 (fr) * 1992-10-22 1994-04-28 The New York Blood Center, Inc. Recyclage d'enzymes utilisees dans la conversion d'erythrocytes des sous-types a, b et ab

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
See also references of WO9623869A1 *
ZHU A ET AL: "Cloning and functional expression of a cDNA encoding coffee bean alpha-galactosidase." GENE, MAR 25 1994, 140 (2) P227-31, NETHERLANDS, XP002062116 *
ZHU, ALEX GOLDSTEIN, JACK: "High-level expression and purification of coffee bean alpha-galactosidase produced in the yeast Pichia pastoris." ARCH BIOCHEM BIOPHYS, DEC 1 1995, 324 (1) P65-70, UNITED STATES, XP002062117 *

Also Published As

Publication number Publication date
EP0807165A4 (fr) 1998-07-15
AU4772596A (en) 1996-08-21
WO1996023869A1 (fr) 1996-08-08
CA2211417A1 (fr) 1996-08-08
JPH10513057A (ja) 1998-12-15

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