弹性蛋白酶基因的克隆及在毕赤酵母中的表达

本研究以一株产弹性蛋白酶的铜绿假单胞菌基因组DNA为模板，经PCR扩增得到的弹性蛋白基因，与GenBank中的序列对比发现同源性为99%。将弹性蛋白酶基因连入到表达载体pPIC3.5K中，经过酶切和测序鉴定证实弹性蛋白酶基因已插入到载体启动子下游，成功构建了质粒pPIC3.5K/PAE。将pPIC3.5K/PAE线性化，通过电转化将目的基因转入毕赤酵母KM71中，利用MD培养基筛选到近400个转化子，再经G418抗性的筛选，获得48株含高拷贝的重组毕赤酵母转化子并用PCR和弹性蛋白平板验证。经过甲醇诱导表达得到高表达的重组酵母菌株，酶活为1060U/mL是出发菌株的26倍。本研究成功克隆到铜绿假单胞菌弹性蛋白酶基因,为实现活性弹性蛋白酶的高效表达奠定了基础。

Cloning of Elastase Coding Sequence and Its Expression in P. pastoris

In this study, the genome DNA of a strain of Pseudomonas aeruginosa was used as the template, via PCR, a 1.67kb product was obtained. We discovered the nucleotide sequence of the cloned DNA shared 99% homology with lasB from P. aeruginosa PA01 (GenBank accession no. M19472). Then the correct plasmid was digested with BamHI and EcoRI and ligated into pPIC3.5K with the same enzymic sites, generating pPIC3.5K/PAE. The insertion was identified by restriction analysis and sequencing.pPIC3.5K/PAE was linearized with Sac I, and electroporated into P. pastoris KM71. Nearly 400 transformants were selected on the MD agar plates，and high-copy transformant weas screened by YPD plates containing G418.Transformant stains were further confirmed by genome PCR and casein-MM plate. After expression of recombinant elastase in shaking flask by methanol induction, we found the size of recombinant elastase was similar to that of the native elastase (approximately 34 kDa) by SDS-PAGE analysis. The highest expression level of the recombinant elastase obtained is approximately 400 mg/L and the specific activity of the recombinant elastase was 1060 U/ml, which was approximately 26-fold higher than that of elastase obtained from P. aeruginosa. The Pseudomonas aeruginosa elastase gene was successfully cloned in this study, which has laid a foundation for high level expression of elastase.