Expression, purification, and characterization of recombinant alpha-N-acetylgalactosaminidase produced in the yeast Pichia pastoris

Protein Expression and Purification
A ZhuJ Goldstein

Abstract

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, We...Continue Reading

Citations

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