Chemistry: molecular biology and microbiology – Spore forming or isolating process
Reexamination Certificate
1993-08-30
2001-12-11
Achutamurthy, Ponnathapu (Department: 1652)
Chemistry: molecular biology and microbiology
Spore forming or isolating process
C435S252300, C435S320100, C435S208000, C536S023200
Reexamination Certificate
active
06329191
ABSTRACT:
TECHNICAL FIELD
The invention is directed to recombinant production of coffee bean &agr;-galactosidase for use in modifying human red blood cells. More specifically, the invention concerns a recombinant enzyme useful in the conversion of Type B red blood cells to cells having the physiological effect of Type O.
BACKGROUND ART
The desirability of obtaining “universal donor”-type red blood cells for transfusion is well recognized. Red blood cells of the “O” type—i.e., lacking both the “A” and “B” antigenic determinants—are suitable. Treatment of the Type A antigenic determinant with N-acetylgalactoseaminidase to destroy its antigenicity has been disclosed in U.S. Pat. No. 4,609,627. Of more relevance to the present invention, the use of coffee bean &agr;-galactosidase for the conversion of Type B antigen to the antigenic equivalent of Type O has also been described by Goldstein in U.S. Pat. Nos. 4,330,619 and 4,427,777. Also describing this work are papers by Lenny, L. L., et al.,
Blood
(1991) 77:1383-1388, and by Goldstein, J., et al.,
Science
(1982) 215:168-170.
In the work by the Goldstein group, it was found necessary to equilibrate the erythrocytes to a pH of about 5.7-5.8 before contacting the erythrocytes with the &agr;-galactosidase. This equilibration was found to prevent the hemolysis noted in attempts to remove the terminal galactose residue from the B determinant using conditions similar to those reported by Zarnitz, M. L., et al.,
J Am Chem Soc
(1960) 82:3953-3957, and by Harpaz, N., et al.,
Arch Biochem Biophys
(1975) 170:676-683. These earlier conversions had been conducted at a pH of about 5.
The coffee bean &agr;-galactosidase utilized in the foregoing studies in U.S. Pat. Nos. 4,330,619 and 4,427,777 is apparently equivalent in purity to the commercially available &agr;-galactosidase (EC 3.2.1.22) marketed by Boehringer Mannheim. Further purification by undisclosed methods may have been conducted by Goldstein (1982) supra and Lenny (1991) supra. The molecular weight of coffee bean &agr;-galactosidase was reported to be 26 kD by Barham, D., et al.,
Phytochem
(1971) 10:1759-1763. The &agr;-galactosidase can be purified using affinity chromatography with a substrate or substrate analog, as described by Courtois, J. E., et al.,
Meth Enzymol
(1966) 8:565-571, and by Harpaz, N., et al.,
Biochem Biophys Acta
(1974) 341:213-221. As shown hereinbelow, the commercial preparation of coffee bean &agr;-galactosidase available is, in fact, an impure mixture of at least four proteins; only a vanishingly small amount of this preparation has the molecular weight 26 kD. In general, the readily detectable proteins in the mixture have molecular weights of 68 kD (40%), 40 kD (35%) and 36 kD (10%); the 68 kD protein is BSA added as a stabilizer. The 40 kD protein has the &agr;-galactosidase activity and corresponds in molecular weight to the deduced amino acid sequence as described below.
The work of Goldstein et al. cited above indicates that coffee bean &agr;-galactosidase is the enzyme of choice for the conversion of B antigen on erythrocytes to that consistent with universal donor erythrocytes. Although it is thus clear that coffee bean &agr;-galactosidase is desirable for this conversion, practical sources for pure preparations of this enzyme for such use have not been available.
The genes or cDNAs encoding &agr;-galactosidases from other sources have been retrieved and reported. These enzymes are not as effective for the degalactosylation of B antigen. Most closely related to the &agr;-galactosidase of the invention is the enzyme from the legume guar (
Cyamopsis tetragonaloba
) seed. (See Hughes, S. G., et al.,
Plant Mol Biol
(1988) 11:783-789; Overbeeke, N., et al.,
Plant Mol Biol
(1989) 13:541-550; and PCT Application WO87/07641.) The cDNA encoding the guar &agr;-galactosidase has been expressed and the protein secreted from
B. subtilis
, as reported by Overbeeke, N.,
Applied Environment Microbiol
(1990) 1429-1434. This enzyme, however, has the disadvantage of a considerably lower pH optimum than that of coffee bean &agr;-galactosidase. Retrieval of &agr;-galactosidase-encoding cDNA from coffee bean by methods analogous to those used for the guar enzyme cDNA is not possible, since in the guar seed &agr;-galactosidase is synthesized in the aleurone layer during germination and an aleurone layer is lacking in coffee beans. Accordingly, the procedures followed in obtaining guar messenger RNA encoding &agr;-galactosidase cannot be followed in coffee bean.
Other sources of &agr;-galactosidase also are known, and the genes or cDNAs have been cloned in some cases. The gene encoding the &agr;-galactosidase of yeast has been reported by Liljestrom, P. L.,
Nucleic Acids Res
(1985) 13:7257-7269; Sumner-Smith, M., et al.,
Gene
(1985) 36:333-340. The DNA encoding the enzyme from
E. coli
is described by Liljestrom, P. L., et al.,
Nucleic Acids Res
(1987) 15:2213-2220. Human &agr;-galactosidase A has been implicated in Fabry disease, and cDNA encoding this protein has been isolated (Calhoun, D. H., et al.,
Proc Natl Acad Sci USA
(1985) 83:7364-7368; Biship, D. F., et al.,
Proc Natl Acad Sci USA
(1986) 83:4849-4853). A genomic clone containing the promoter for this gene was reported by Quinn, M., et al.,
Gene
(1987) 58:177-188, and the recombinant enzyme has been suggested as a treatment for Fabry disease, as set forth in PCT Application WO90/11353 and U.S. Pat. No. 5,179,023.
None of these genes appears suitable for the production of &agr;-galactosidase for use in the conversion of B erythrocytes, since this range of enzymes has considerable variance in substrate specificity and differences in pH optima. For example, Overbeeke et al. (1987 PCT Application WO87/07641) demonstrate that &agr;-Gals of plant origin (guar, fenugreek, lucerne, and coffee beans) are able to reduce the galactose content of galactomannans, whereas microbial (
Aspergillus niger, Saccharomyces carlsbergensis
, and
Escherichia coli
) &agr;-Gals lack this enzymatic capability. Although it has been shown that &agr;-galactosidase from
Clostridium sporogenes
(Dybus, S.,
Transfusion
(1983) 23:244-247) and from soybeans (Harpaz, N., et al.,
Eur J Biochem
(1977) 77:419-426) can remove the terminal galactose from B-antigen, these enzymes are more complex and the pH optima appear to be too low to effect the desired conversion on erythrocytes, since at low pH values, hemolysis occurs.
Thus, the present invention provides, for the first time, a practical source for purified and isolated coffee bean &agr;-galactosidase required for the conversion of B-type red blood cells to red blood cells capable of serving as universal donors.
DISCLOSURE OF THE INVENTION
The invention is directed to the recombinant production of coffee bean &agr;-galactosidase (coffee bean &agr;-Gal). The production of this protein in recombinant form permits the practical production of sufficient quantities of pure enzyme for use in effecting the production of universal donor red blood cells.
Thus, in one aspect, the invention is directed to DNA encoding coffee bean &agr;-galactosidase having the amino acid sequence shown in
FIG. 1
herein or &agr;-galactosidase encoded by the allelic variants of its encoding DNA. The invention is also directed to expression systems for the production of this protein and to methods for its production using these systems.
In other aspects, the invention is directed to methods to manufacture universal donor red blood cells by treating red blood cells containing Type B antigen with the recombinant enzyme of the invention, to antisense and triple helix-forming oligomers, and to antibodies specific for coffee bean &agr;-Gal.
REFERENCES:
patent: 4330619 (1982-05-01), Goldstein
patent: 4427777 (1984-01-01), Goldstein
patent: 4609627 (1986-09-01), Goldstein
patent: 5082778 (1992-01-01), Overbeeke et al.
patent: 5179023 (1993-01-01), Calhoun et al.
patent: 5296365 (1994-03-01), Overbeeke et al.
patent: WO87/07641 (1987-12-01), None
patent: WO90/11353 (1990-10-01), None
Wieder et al., “Enzym
Clements David E.
Ivy John M.
Achutamurthy Ponnathapu
Hawaii Biotechnology Group, Inc.
Morrison & Foerster / LLP
Tung Peter P.
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