New York Blood Center, Inc.

Jack Goldstein

Alex Zhu

Lin Leng

Rosa S Hurst

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

Recombinant &agr;-N-acetylgalactosaminidase enzyme and cDNA encoding said enzyme

The present invention relates to purified and isolated nucleic acid encoding the endo-.beta.-galactosidase from Flavobacterium keratolyticus (referred to as 'ENDO-A'), and to purified ENDO-A protein. The endo-.beta.-galactosidase of the invention may be used in a process which enzymatically de-antigenizes human erythrocytes bearing A.sub.1 antigen. The resulting erythrocytes may be transfused into individuals who would be otherwise unable to tolerate a transfusion of type A.sub.1 blood.

Endo-.beta.-galactosidase

This invention relates to an improved method of purifying the enzyme .alpha.-N-acetylgalactosaminidase from avian liver, as well as to the purified enzyme. The enzyme is capable of removing A antigens from the surface of cells in blood products. The method of purifying the enzyme is simple and time efficient, and lends itself to large scale production.

Purification of .alpha.N-acetylgalactosaminidase

Type O erythrocytes are produced from certain subtypes of A erythrocytes or type AB erythrocytes by contacting the same following equilibration of a pH of 5.6-5.8 with an .alpha.-N-acetylgalactosaminidase, preferably obtained from an avian liver, for periods sufficient to convert the A antigen in the erythrocyte to the H antigen. Following removal of the enzyme, the erythrocyte is re-equilibrated to a pH of 7.2-7.4. As a result, there is obtained O type erythrocytes characterized by a 60 to 90 percent ATP level based on the level of ATP in naturally occurring O or AB erythrocytes. Beginning with certain A cells one obtains synthetic O erythrocytes characterized by a terminal .alpha.-fucose moiety, O antigenicity, and the absence of A antigenicity. Beginning with A.sub.2 B erythrocytes, one obtains B erythrocytes by the same process characterized by the absence of A antigenicity, greater H antigenicity than naturally occurring A.sub.2 B cells, the presence of B antigenicity and the aforedescribed ATP levels.

Enzymatic conversion of certain sub-type A and AB erythrocytes

Transfusable type O erythrocytes free of P1 antigenicity which are produced by an alpha-galactosidase conversion of type B antigen to type H antigen. The resulting erythrocytes retain a high level of ATP and 2,3 DPG.

Enzymatic conversion of red cells for transfusion

A composition comprising type O erythrocyte free of P.sub.1 antigenicity; a method of converting erythrocytes of the B antigen type to erythrocytes of the H-antigen type which comprises:

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New York, NY, United States of America

Jack Goldstein