Overexpression of a small medicinal peptide from ginseng in the yeast Pichia pastoris.
A medicinal peptide, Gsp, which was initially extracted from the traditional medicinal herb ginseng, has potential use as a drug against diabetes. Gsp is a low molecular weight protein that we have secreted in a recombinant form from the yeast Pichia pastoris. A DNA fragment encoding four copies of the Gsp protein each separated by a basic amino acid was synthesized and inserted into the P. pastoris expression vector plasmid pPIC9. After electroporation of the resulting vector, pPIC9-Gsp, into the yeast, transformants were selected. Recombinant pre-Gsp secreted from P. pastoris had a molecular weight of 5.9 kDa and mature recombinant Gsp had a primary structure indistinguishable from native Gsp. After optimization of the culturing process, the yield of pre-Gsp reached 800 mg/L in the clarified broth. A continuous batch fermentation process was developed that allowed the same population of cells to be reutilized five times without loss of expression level. This continuous culturing process resulted in a substantial saving of both time and cost in pharmaceutical production and should be applicable to the production of other recombinant proteins in P. pastoris.
Yan
Yajun
Y
Department of Pharmaceutical Engineering, College of Chemical Engineering, Beijing University of Chemical Technology, Beijing 100029, People's Republic of China.
Chen
Jingchun
J
Li
Jun
J
D000818:Animals; D001709:Biotechnology / Q000379:methods; D001786:Blood Glucose / Q000032:analysis / Q000187:drug effects; D003921:Diabetes Mellitus, Experimental / Q000188:drug therapy; D005285:Fermentation; D015870:Gene Expression; D005822:Genetic Vectors; D008297:Male; D009842:Oligopeptides / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification / Q000494:pharmacology; D005894:Panax / Q000737:chemistry*; D010843:Pichia / Q000378:metabolism*; D010940:Plant Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification / Q000494:pharmacology; D010957:Plasmids / Q000235:genetics; D051381:Rats; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000494:pharmacology
D001786:Blood Glucose; D009842:Oligopeptides; D010940:Plant Proteins; D011994:Recombinant Proteins