High-level expression, purification, and characterization of porcine somatotropin in Pichia pastoris.
Porcine somatotropin (pST) significantly improves the growth rate, carcass composition, and growth efficiency of pigs while reducing feed consumption and fat deposition. Pichia pastoris was used as a host to efficiently express the pST gene in this study. Up to 90% of the recombinant protein was secreted into the culture medium, yielding about 900 mg/L rpST in shake-flask cultures. SDS-PAGE and Western blot analyses showed that rpST migrated as a single band with a molecular weight of approximately 25 kDa, and had the same immunoreactivity as native pST. The culture supernatant of our rpST expression strain, X-33/pPICZalphaA-pST/9, was purified to greater than 95% homogeneity with 71.4% recovery using ammonium sulfate precipitation, Sephadex G-25 Fine desalting, and Q Sepharose High Performance Ion Exchange chromatography. MALDI-TOF-MS demonstrated a molecular mass of 21,771Da for rpST, close to its predicted size. Isoelectric focusing electrophoresis results from three batches of purified rpST consistently showed a pI value between 4.55 and 5.2. Purified rpST was able to promote Nb2 cell proliferation and reduce feed intake of crossbred gilts, a type of pig breed, with no decrease in body weight gain when administered by injection. These results indicate that the P. pastoris expression system will be useful for production of bioactive rpST at commercially relevant levels.
Ouyang
Jing
J
State Key Laboratory for Biocontrol, Biopharmaceutical Center, Sun Yat-Sen University, 135 Xingang West Road, Guangzhou 510275, PR China.
Wang
Jinwen
J
Deng
Riqiang
R
Long
Qingxin
Q
Wang
Xunzhang
X
D000818:Animals; D002455:Cell Division / Q000187:drug effects; D045744:Cell Line, Tumor; D004435:Eating; D005822:Genetic Vectors / Q000235:genetics; D006031:Glycosylation; D013006:Growth Hormone / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification* / Q000494:pharmacology; D008223:Lymphoma / Q000473:pathology; D010843:Pichia / Q000235:genetics*; D051381:Rats; D013552:Swine
D013006:Growth Hormone