Abstract: Crustins are series of antibacterial peptides rich in cysteine from crustaceans, which are important immune factors for nonspecific immune mechanism of crustaceans， and their biological functions are attributed to the mature peptide region near the C terminal. A codon-optimized sequence corresponding to the swimming crab (Portunus trituberculatus) PtCrustin2 mature peptide cDNA was designed and synthesized based on the preferential codon usage of Pichia pastoris. This synthesized cDNA fragment (smPtCrustin2) contained Xho I restriction site and Kex2 signal cleavage site at its 5′ end, and Xba I restriction site and 6 his tag at its 3′ end. This target fragment was ligated to pPICZαA to construct a recombinant expression vector pPICZαA-smPtCrustin2. Subsequently the linearized pPICZαA-smPtCrustin2 was transformed into competent Pichia X-33 cells by electroporation. The yeast transformants harboring multi-copy gene insertions were screened using plate containing high concentration of zeocin. The target protein was induced with 0.5% methanol at 28℃ and 250 r/min. The recombinant smPtCrustin2 was purified by immobilized metal affinity chromatography (IMAC). Tricine-SDS-PAGE analysis showed that the molecular weight of purified recombinant smPtCrustin2 was about 9.6 ku, and an about 20 ku-dimer was also found. Western Blot analysis further confirmed that the purified product was the expected target protein. The recombinant smPtCrustin2 with an expression yield of 22.4 mg/L was obtained by the P. pastoris yeast expression system established in this study.

Key words: swimming crab, PtCrustin2 mature peptide, codon optimization, Pichia pastoris, recombinant DNA expression