Expression of black carp growth hormone in pichia pastoris

In order to amplify the cDNA of black carp growth hormone,the primers were designed according to the GenBank sequence of black carp growth hormone gene(AF389238).The black carp growth hormone(bcGH) cDNA was oriently inserted into pPIC3.5k which contains AOXI promoter and Pichia wild-type gene coding for histidinol dehydrogenase.The recombinant plasmid pPIC3.5k-bcGH was linearized by restriction endonucleases Sal I,then transformed into HIS4 mutant yeast GS115 by electroporation,the recombinant growth hormone yeasts were certified by the screening of G418 and sequencing.SDS-PAGE and ELISA analysis showed that bcGH gene expression product appeared in intracellular solution.Under the optimization of fermentation conditions,the recombinant protein can be expressed in induction time 24 h and pH 4.5~7.0.Under the optimized conditions of 30 ℃,pH 6.5,fermentation time 72 hours and induced concentration 0.5%,the yield is 893 mg/L.