Cloning and expression of fox growth hormone gene in Pichia pastoris.
- Author:
Wei LI
1
;
Xiujin LI
;
Fei ZHONG
;
Huijun JIN
;
Min XIE
;
Yuzhi LIU
;
Longfei LIU
;
Qingjie SU
Author Information
1. Department of Basic Veterinary Medicine, College of Animal Science and Technology, Agricultural University of Hebei, Baoding 071001, China.
- Publication Type:Journal Article
- MeSH:
Animals;
Base Sequence;
Cloning, Molecular;
DNA, Complementary;
genetics;
Electroporation;
Foxes;
genetics;
Genetic Vectors;
genetics;
Growth Hormone;
biosynthesis;
genetics;
Molecular Sequence Data;
Pichia;
genetics;
metabolism;
Recombinant Proteins;
biosynthesis;
genetics
- From:
Chinese Journal of Biotechnology
2009;25(10):1470-1476
- CountryChina
- Language:Chinese
-
Abstract:
To prepare recombinant fox growth hormone (fGH), we amplified its cDNA from silver fox pituitary tissue by RT-PCR and cloned into yeast shuttle vector pPIC9K down stream of a-factor signal peptide sequence by SnaB I and Not I restriction sites. The recombinant secretion vector pPIC9K/fGH, linearized by Sal I, was transformed into histidine-deficient Pichia pastoris strain GS115 by electroporation. We selected His+ -transformed methylotropic (His+, Mut+) yeast using histidine-absent medium containing dextrose (MD) or methanol (MM) as the only carbon source, and then screened the recombinant GS115 with multi-copy fGH genes by G418. The secretive expression of fGH was performed under the induction of methanol in shaking flask culture. The results showed that the fGH cDNA sequence amplified in this paper was basically in consistence with the published in GenBank. We achieved the secretive expression of recombinant fGH identified by SDS-PAGE and Western blotting. The fGH expression level was 119 mg/L, accounted for 34% of total proteins in fermentation medium.
