Expression of the alpha-galactosidase from Cyamopsis tetragonoloba (guar) by Hansenula polymorpha.
The methylotrophic yeast Hansenula polymorpha, a host organism for the production of heterologous proteins, has been applied to produce the alpha-galactosidase from the plant Cyamopsis tetragonoloba (guar). The yeast/Escherichia coli shuttle expression vector used is based on the origin of replication of the endogenous 2 microns plasmid of Saccharomyces cerevisiae and the LEU2 gene of S. cerevisiae for selection in H. polymorpha. In the expression vector, the alpha-galactosidase is controlled by the methanol-regulated promoter from the methanol oxidase gene, MOX, of H. polymorpha. The signal sequence of SUC2 (invertase) from the yeast S. cerevisiae, was used to ensure secretion of the alpha-galactosidase enzyme. After transformation and stabilization, the expression vector was stably integrated in the genome. The active alpha-galactosidase enzyme was efficiently secreted (greater than 85%) and after methanol induction, the expression level was 42 mg/l. Amino-terminal sequencing of the purified alpha-galactosidase enzyme synthesized by H. polymorpha showed that the S. cerevisiae invertase signal sequence was correctly processed by H. polymorpha. The secreted alpha-galactosidase was glycosylated and had a sugar content of 9.5%. The specific activity of the alpha-galactosidase produced by H. polymorpha was 38 U mg-1 compared to 100 U mg-1 for the guar alpha-galactosidase. Deglycosylation of the H. polymorpha alpha-galactosidase restored the specific activity completely.
Fellinger
A J
AJ
Unilever Research Laboratorium, Vlaardingen, The Netherlands.
Verbakel
J M
JM
Veale
R A
RA
Sudbery
P E
PE
Bom
I J
IJ
Overbeeke
N
N
Verrips
C T
CT
D001483:Base Sequence; D015152:Blotting, Northern; D015139:Blotting, Southern; D015153:Blotting, Western; D003001:Cloning, Molecular; D003470:Culture Media; D004271:DNA, Fungal / Q000737:chemistry; D007887:Fabaceae / Q000201:enzymology*; D015966:Gene Expression Regulation, Fungal; D005822:Genetic Vectors; D006031:Glycosylation; D008969:Molecular Sequence Data; D010843:Pichia / Q000201:enzymology / Q000235:genetics*; D010946:Plants, Medicinal; D010957:Plasmids; D011401:Promoter Regions, Genetic; D012331:RNA, Fungal / Q000737:chemistry; D015183:Restriction Mapping; D012639:Seeds / Q000201:enzymology*; D014170:Transformation, Genetic; D000519:alpha-Galactosidase / Q000096:biosynthesis* / Q000235:genetics
D003470:Culture Media; D004271:DNA, Fungal; D012331:RNA, Fungal; D000519:alpha-Galactosidase