三疣梭子蟹PtCrustin2抗菌肽基因优化及其在毕赤酵母中高效表达

Gene optimization for swimming crab PtCrustin2 antimicrobial peptide and its efficient expression in Pichia pastoris

Crustin是一系列富含半胱氨酸的甲壳动物来源的抗菌肽,是甲壳动物非特异性免疫机制中的重要免疫因子,其生物学功能由C端的成熟肽区域决定。参考三疣梭子蟹( Portunus trituberculatus ) PtCrustin2成熟肽的cDNA序列,根据毕赤酵母( Pichia pastoris )的密码子偏爱性对其相关密码子进行优化,合成5′端含 Xho Ⅰ限制性位点和Kex2信号肽酶切位点、3′端含 Xba Ⅰ限制性位点和6×his标签的目的基因“ smPtCrustin2 ”;以pPICZαA为表达载体,构建重组表达载体pPICZαA- smPtCrustin2,将其转入毕赤酵母X-33细胞后,通过含高浓度博来霉素的抗性平板筛选高拷贝甲醇利用快速型酵母转化子;筛选获得的酵母转化子在28 ℃、250 r/min条件下,使用0.5%甲醇诱导表达目的蛋白,并通过固化金属离子亲和层析(IMAC)对其进行分离纯化。Tricine-SDS-PAGE分析显示:纯化的重组体smPtCrustin2的分子量约为9.6 ku,并形成分子量约20 ku的二聚体。Western Blot分析进一步证明了纯化产物为预期的目的蛋白。建立的毕赤酵母表达系统获得了表达量为22.4 mg/L的重组体smPtCrustin2。

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.