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 from chicken liver has been used in seroconversion of human erythrocytes. Blood group A, defined by the terminal alpha-linked N-acetylgalactosamine, can be cleaved in vitro by alpha NAGAL, resulting in the underlying penultimate blood group H (O) epitope structure. In order to produce sufficient quantities of recombinant alpha NAGAL (r alpha NAGAL) for such studies, we expressed the cDNA encoding chicken liver alpha NAGAL in Pichia pastoris, a methylotrophic yeast strain. The alpha NAGAL coding sequence was cloned into the EcoRI site of the vector pPIC 9 such that the protein was in the same reading frame as the secretion signal of yeast alpha-mating factor derived from the vector. After P. pastoris transformation, colonies were screened for high-level expression of r alpha NAGAL based on enzyme activity. As a result of methanol induction of high-density cell cultures in a fermentor, enzymatically active r alpha NAGAL was produced and secreted into the culture medium. The recombinant enzyme was purified over 150-fold by chromatography on a cation exchange column followed by an affinity column. Its homogeneity was confirmed by Coomassie blue-stained SDS-PAGE, Western blot, and N-terminal sequencing. The purified r alpha NAGAL has a molecular mass of approximately 50 kDa while its native counterpart has a molecular mass of 43 kDa. This discrepancy in size was eliminated by endoglycosidase treatment, suggesting that the recombinant protein was hyperglycosylated by the host P. pastoris cells. r alpha NAGAL was further characterized in terms of specific activity, pH profile, kinetic parameters, and thermostability by comparing with alpha NAGAL purified from chicken 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 seroconversion studies.
Zhu
A
A
Lindsley F. Kimball Research Institute of the New York Blood Center, New York 10021, USA.
Monahan
C
C
Wang
Z K
ZK
Goldstein
J
J
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D002645:Chickens; D018076:DNA, Complementary / Q000737:chemistry; D006596:Hexosaminidases / Q000737:chemistry / Q000302:isolation & purification*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008099:Liver / Q000201:enzymology; D008969:Molecular Sequence Data; D010843:Pichia; D048809:alpha-N-Acetylgalactosaminidase
D018076:DNA, Complementary; D006596:Hexosaminidases; C482722:NAGA protein, human; D048809:alpha-N-Acetylgalactosaminidase