Abstract: Based on the known insulin-like androgenic gland hormone (IAG) gene in Portunus trituberculatus nucleotide sequence, the ORF region of Pt-IAG was amplified, liganded into different prokaryotic vectors, including pET-22b, pET-28a and pET-28a-sumo, and then transformed into Escherichia coli BL21 (DE3) and Rosetta (DE3) strains, respectively. The His-tag labeled IAG recombinant fusion protein was induced by isopropyl-β-D-thiogalactoside (IPTG), and the SDS-PAGE found that only the pET-28a-sumo-IAG could be detected, and the IAG transformed into the Rosetta (DE3) strain was highly expressed. Further exploration of the condition of this Rosetta-pET-28a-sumo-IAG system showed that the IAG recombinant fusion protein was most efficiently expressed when induced by 0.6 mmol/L of IPTG at 16 ℃ for 16 h. The fusion protein was mainly expressed in soluble form and was further purified by HisTrap HP column. Western Blot analysis showed that the fusion protein could be recognized by mouse anti-His-tag. The purified protein was further analyzed by mass spectrometry, and two peptides could be matched to the B chain and A chain, respectively, which indicated that the recombinant fusion protein was successfully obtained.

Key words: Portunus trituberculatus, insulin-like androgenic gland hormone gene, prokaryotic expression, mass spectrometry determination