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 al pha-galactose residues from oligosaccharide chains on blood group B re d cells, thus generating group O cells. Such enzymatically converted r ed cells not only maintain full erythrocyte integrity and viability in vitro, but also demonstrate immune tolerance and a normal life span i n vivo, In order to produce large quantities of recombinant alpha-gala ctosidase for use in the study of blood-type conversion, we subcloned the cDNA coding for coffee bean alpha-galactosidase into the EcoRI sit e of the vector pPIC9 in order to express the enzyme in Pichia pastori s, a methylotrophic yeast strain. After P. pastoris transformation, co lonies 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 we re optimized. Under the conditions applied, biologically active alpha- galactosidase was produced and secreted into the culture medium at a l evel of approximately 0.4 g per liter of the fermenter culture. The pr otein was purified to apparent homogeneity by a simple chromatography procedure, as suggested by a single band of 41 kDa on sodium dodecyl s ulfate-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 rec ombinant alpha-galactosidase used for blood type conversion. (C) 1995 Academic Press, Inc.