人β防御素3与植物巴西甜蛋白基因密码子的优化及其在毕氏酵母中的表达

Optimization mosaic gene of human β defensin 3 and plant des-pGlu1-Brazzein and its expression in Pichia pastoris

根据人β防御素3和植物甜蛋白des-pGlu1-Brazzein的氨基酸序列,按照酵母密码子的偏爱性,合成了14段末端具有粘合位点的核苷酸序列,经连接、PCR扩增,使人β防御素3和植物甜蛋白des-pGlu1-Brazzein的编码序列通过1段序列(含凝血酶切割位点)连接成为嵌合基因,将其插入到pPIC9K载体中,构建重组表达载体pPIC9K-hBD3-Bra.SalⅠ,BglⅡ单酶切后,分别电击转化到毕赤酵母GS115中,构建野生型和乙醇氧化酶缺陷型酵母工程菌株GS115-pPIC9K-hBD3-Bra.筛选鉴定后,以体积分数为0.5%的甲醇进行诱导,缺陷型工程菌株表达的目的蛋白约占上清蛋白的90%,纯度较高,野生型工程菌株表达的目的蛋白约占上清蛋白的57.8%.

According to the sequences of human β defensin 3 and plant sweet-tasting protein despGlul-Brazzein, fourteen pairs of oligoneucleotide with cosmid site were synthesized using yeasty biased codons. After linkage and PCR, the DNA sequence of human β defensin 3 and plant des-pGlu1- Brazzein were incorporated together with the sequences coding thrombin cleavage site between them. This recombinant gene sequence was inserted into pPIC9K which resulted in recombinant expression vector pPIC9K-hBD3-Bra. Linearized by Sal Ⅰ or Bgl Ⅱ ,the vectors were transformed into Pichia pastoris strain GS115 by electro shock to produce wild type and alcohol oxidase-defective expression strain respectively. Identified by G418 and PCR, the strain was induced by 0.5% methanol. The results showed that the target protein accounted for 57.8% and 90% of total protein in the supernatant for wild type and alcohol oxidase-defective strain respectively.