کد مقاله | کد نشریه | سال انتشار | مقاله انگلیسی | نسخه تمام متن |
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2020594 | 1069193 | 2012 | 6 صفحه PDF | دانلود رایگان |

IGF-1 plays a key role in development, growth, and metabolism in teleost. Recombinant fish IGF-1 may be a useful tool for both theoretical research and aquaculture applications. However, using the Escherichia coli expression system has several drawbacks for producing quality fish IGF-1 protein. To explore the yeast expression system for generating fish IGF-1 protein, the cDNA coding for the mature orange-spotted grouper IGF-1 peptide without signal peptide and E domain was cloned into the secreting expression organism Pichia pastoris. Tricine–SDS–PAGE and western blotting analysis of culture medium from methanol-induced expression yeast clones demonstrated that the rgIGF-1 was secreted into the culture medium, had a molecular weight of 8.7 kDa. The production peaked at 24 h of induction and the optimal pH for expression was 5.0. The recombinant protein was purified using a combined ammonium sulfate precipitation with Ni2+ affinity chromatography. Finally, 17.9 mg of the protein was obtained from 420 ml of the culture supernatant and the purity was about 92.4%. Bioactivity of the rgIGF-1 was confirmed by the ability to stimulate proliferation of embryo cell line of grouper (GP cell line) and MFC-7 cell. The present results suggest that the Pichia pastoris expression system can be used to produce a functional rgIGF-1 for both research and aquaculture application.
► Recombinant grouper IGF-1 was expressed with Pichia pastoris system.
► The production peaked at 24 h of induction and the optimal pH for expression was 5.0.
► The yield of the recombinant protein was 42.8 mg with the purity of 92.4% from 1 L of the culture supernatant.
► The rgIGF-1 can stimulate proliferation of embryo cell line of grouper (GP cell line) and MFC-7 cell.
Journal: Protein Expression and Purification - Volume 84, Issue 1, July 2012, Pages 80–85