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. (c) 2012 Elsevier Inc. All rights reserved.