High-level expression and purification of coffee bean alpha-galactosidase produced in the yeast Pichia pastoris

alpha-Galactosidase isolated from coffee beans cleaves the terminal alpha-galactose residues from oligosaccharide chains on blood group B red cells, thus generating group O cells. Such enzymatically converted red cells not only maintain full erythrocyte integrity and viability in vitro, but also demonstrate immune tolerance and a normal life span in vivo, In order to produce large quantities of recombinant alpha-galactosidase for use in the study of blood-type conversion, we subcloned the cDNA coding for coffee bean alpha-galactosidase into the EcoRI site of the vector pPIC9 in order to express the enzyme in Pichia pastoris, a methylotrophic yeast strain. After P. pastoris transformation, colonies were screened for high-level expression of alpha-galactosidase, based on enzyme activity, In order to increase enzyme production, the growth conditions in the shake flask culture and fermenter culture were optimized. Under the conditions applied, biologically active alpha-galactosidase was produced and secreted into the culture medium at a level of approximately 0.4 g per liter of the fermenter culture. The protein was purified to apparent homogeneity by a simple chromatography procedure, as suggested by a single band of 41 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, Its homogeneity was further confirmed by chromatofocusing and N-terminal sequencing, P. pastoris appears to be the choice as host for the large-scale production of recombinant alpha-galactosidase used for blood type conversion. (C) 1995 Academic Press, Inc.