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 fermentor 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 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. pastoris appears to be the choice as host for the large-scale production of recombinant alpha-galactosidase used for blood type conversion.
Zhu
A
A
Lindsley F. Kimball Research Institute, New York Blood Center, New York 10021, USA.
Monahan
C
C
Zhang
Z
Z
Hurst
R
R
Leng
L
L
Goldstein
J
J
D000595:Amino Acid Sequence; D001483:Base Sequence; D003069:Coffee / Q000201:enzymology* / Q000235:genetics; D008969:Molecular Sequence Data; D010843:Pichia / Q000235:genetics / Q000254:growth & development; D011994:Recombinant Proteins / Q000096:biosynthesis; D012639:Seeds / Q000201:enzymology* / Q000235:genetics; D012641:Selection, Genetic; D014170:Transformation, Genetic; D000519:alpha-Galactosidase / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification
D003069:Coffee; D011994:Recombinant Proteins; D000519:alpha-Galactosidase