High-Level Expression and Purification of Coffee Bean α-Galactosidase Produced in the YeastPichia pastoris

α-Galactosidase isolated from coffee beans cleaves the terminal α-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 viabilityin vitro,but also demonstrate immune tolerance and a normal life spanin vivo.In order to produce large quantities of recombinant α-galactosidase for use in the study of blood-type conversion, we subcloned the cDNA coding for coffee bean α-galactosidase into theEcoRI site of the vector pPIC9 in order to express the enzyme inPichia pastoris,a methylotrophic yeast strain. AfterP. pastoristransformation, colonies were screened for high-level expression of α-galactosidase, based on enzyme activity. In order to increase enzyme production, the growth conditions in the shake flask culture and fermentor culture were optimized. Under the conditions applied, biologically active α-galactosidase was produced and secreted into the culture medium at a level of approximately 0.4 g per liter of the fermentor 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. pastorisappears to be the choice as host for the large-scale production of recombinant α-galactosidase used for blood type conversion.