EXPRESSION, PURIFICATION, AND CHARACTERIZATION OF RECOMBINANT ALPHA-N-ACETYLGALACTOSAMINIDASE PRODUCED IN THE YEAST PICHIA-PASTORIS

alpha-N-Acetylgalactosaminidase (alpha NAGAL, EC 3.2.1.49) purified fr om chicken liver has been used in seroconversion of human erythrocytes . Blood group A, defined by the terminal alpha-linked N-acetylgalactos amine, can be cleaved in vitro by alpha NAGAL, resulting in the underl ying penultimate blood group H (O) epitope structure. In order to prod uce sufficient quantities of recombinant alpha NAGAL (r alpha NAGAL) f or such studies, we expressed the cDNA encoding chicken liver alpha NA GAL in Pichia pastoris, a methylotrophic yeast strain. The alpha NAGAL coding sequence was cloned into the EcoRI site of the vector pPIC 9 s uch that the protein was in the same reading frame as the secretion si gnal of yeast alpha-mating factor derived from the vector. After P. pa storis transformation, colonies were screened for high-level expressio n of r alpha NAGAL based on enzyme activity. As a result of methanol i nduction of high-density cell cultures in a fermenter, enzymatically a ctive r alpha NAGAL was produced and secreted into the culture medium. The recombinant enzyme was purified over 150-fold by chromatography o n a cation exchange column followed by an affinity column. Its homogen eity was confirmed by Coomassie blue-stained SDS-PAGE, Western blot, a nd N-terminal sequencing. The purified r alpha NAGAL has a molecular m ass of approximately 50 kDa while its native counterpart has a molecul ar mass of 43 kDa. This discrepancy in size was eliminated by endoglyc osidase treatment, suggesting that the recombinant protein was hypergl ycosylated by the host P. pastoris cells. r alpha NAGAL was further ch aracterized in terms of specific activity, pH profile, kinetic paramet ers, and thermostability by comparing with alpha NAGAL purified from c hicken liver. The data presented here suggest that by overexpressing r alpha NAGAL in P. pastoris and purifying with affinity chromatography one can readily obtain the quantity of enzyme needed for seroconversi on studies. (C) 1996 Academic Press, Inc.