[High level secretion expression of porcine somatoropin gene in Pichia pastoris and N-glycosylation analysis of products].
The porcine somatoropin gene was inserted into the Pichia pastoris expression vector of pPICZ alpha A which contains AOX I promoter and alpha-factor signal sequence. The recombinant plasmid of pPICZ alpha A-pST was linearnized by Sac I and transformed into X-33 by electroporation. The multi-copy insert transformants were selected and cultivated in flasks. SDS-PAGE and Western blot analysis showed that PST gene products were observed in the supernants with a little larger molecular weight size than the natural PST's, however, the molecular weight size of the PST gene products in the soluble cellular proteins were identical to the natural PST's. Retransformation of the linearnized pPICZ alpha-pST showed the expression level was improved greatly and rPST has the same antigenicity as natural one. The expressed rPST accumulated up to about 956 mg/L. The N-glycosylation analysis showed rPST had no N-glycosylation.
Ouyang
J
J
State Key Laboratory for Biological Control, Biopharmaceutical Center, Zhongshan University, Guangzhou 510275, China.
Yang
L
L
Long
Q X
QX
Wang
X Z
XZ
Xing
K
K
Wei
P H
PH
D000818:Animals; D015153:Blotting, Western; D017077:Culture Media, Conditioned / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D015870:Gene Expression; D006031:Glycosylation; D013006:Growth Hormone / Q000235:genetics / Q000378:metabolism*; D010843:Pichia / Q000235:genetics* / Q000378:metabolism; D010957:Plasmids / Q000235:genetics; D016133:Polymerase Chain Reaction; D013552:Swine; D014170:Transformation, Genetic
D017077:Culture Media, Conditioned; D013006:Growth Hormone