大西洋庸鲽(Hippoglossus hippoglossus L.)hipposin抗菌肽在毕赤酵母中的表达及其纯化

Expression of Atlantic halibut(Hippoglossus hippoglossus L.) hipposin antimicrobial peptide in Pichia pastoris and its purification

Hipposin是大西洋庸鲽(Hippoglossus hippoglossus L.)组蛋白H2A的N端衍生物,共含51个氨基酸残基,具有抑制革兰氏阳性和阴性菌的活性。根据毕赤酵母密码子偏爱性对编码hipposin的cDNA(hip)进行密码子优化,并在5'端添加信号肽酶kex 2识别位点的编码序列、3'端添加6×His标签编码序列,两个末端分别添加Xho I和Xba I酶切位点,与表达载体pPICZαA构建重组表达载体pPICZαA-hip;电转至毕赤酵母X-33中,经Zeocin筛选以及对酵母基因组DNA的PCR鉴定,获得高拷贝酵母转化子;将其在30℃,250 r/min培养,使用1%的甲醇诱导表达目的蛋白;采用固化金属离子亲和层析(IMAC)纯化目的蛋白。培养96 h时目的蛋白的表达量最高;Western Blot分析显示,表达产物能与His单克隆抗体发生特异性反应;培养上清经纯化后获得高纯度的重组体HIP,但发现部分产物被蛋白酶降解。研究结果为hipposin抗菌肽的扩大制备奠定了基础。

Hipposin is the N-terminal derivative of Atlantic halibut（ Hippoglossus hippoglossus L.） histone H2 A with 51 amino acids,which has a good ability to inhibit gram-positive and gram-negative bacteria. The cDNA encoding hipposin（ hip） was optimized and synthesized with reference to the codon preference of Pichia pastoris and its native cDNA sequence,respectively. In this synthesized fragment,nucleotides encoding recognition site for the enzyme（ Kex2） cutting α-factor signal peptide and 6 × His tag were added to5＇ and 3＇ terminals,respectively,and Xho I and Xba I restriction sites were added to the both ends. This fragment was ligated with pPICZαA to construct the recombinant expression vector pPICZαA-hip. The pPICZαA-hip was transformed into P. pastoris X-33. The yeast transformants containing multicopy gene insertion were selected by using Zeocin,and identified by PCR for yeast genomicDNA.Expression was induced by adding 1% methanol at 30℃ and 250 r / min. Immobilized metal affinity chromatography（ IMAC） was used to purify the target protein. The most appropriate expression time was 96 h. Western Blot analysis demonstrated that the His monoclonal antibody could be specifically bound to expressed products. The highly purified target protein was obtained from the fermentation supernatant,but it was found that a part of the expressed product was hydrolyzed. The present results provide important initial values for preparation of hipposin by recombinant DNA expression.