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Abstract Five primers were synthesized using yeast biased codons, and a 210bp DNA fragment encoding mature peptide of porcine Insulin-like Growth Factor I was amplified by Over Lap Extension Polymerase Chain Reaction. The fragment was then inserted into pPIC9K vector, with the insert being downstream of AOX1 promoter and α-secreting signal peptide. The plasmid was digested with SalI and transformed into HIS4 mutant Pischia pastoris GS115 strain by LiCl. The phenotype of colonies were identified by comparison of the growth performance on MM and MD plates. Two colonies T1, T2 were selected from 4.0 mg/mL G418 plates, followed by induction of pIGF-I expression with 1.5% methanol. SDS-PAGE analysis of supernatant of the P. pastoris GS115 strain revealed an exogenous protein with molecular weight of approximately 7.5 kDa. Dot blot hybridization assay indicated a specific reaction with rabbit polyclonal antibody against the IGF-I protein. The optimal culture condition of T2 was achieved, and 410 mg IGF-I per liter of fluid culture was produced in P. pastoris strain.
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Received: 26 June 2006
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