假单胞茵产弹性蛋白酶基因的克隆与表达

从假单胞菌（Pseudomonassp．）XZG36中克隆弹性蛋白酶基因，构建原核表达载体，实现其在大肠杆菌（Escherichiacoli）中的高效表达，并对表达产物进行酶学性质分析，为微生物发酵生产弹性蛋白酶奠定基础．以假单胞菌基因组DNA为模板，PCR扩增弹性蛋白酶基因，并将其开放阅读框（0RF）克隆至融合表达载体pET30a（＋）进一步IPTG诱导表达；表达产物经His·Bind亲和层析纯化后对弹性蛋白酶进行酶学性质分析．实验成功克隆了弹性蛋白酶基因，DNA基因片段为1672bp、编码497个氨基酸残基的多肽，与预计长度相符合；实现了其在E．coli中的高效表达，表达量约占菌体总蛋白的20％；经SDS-PAGE分析，相对分子质量为48000，与预期的一致；提纯后的表达蛋白SDS-PAGE分析可见单一条带，纯度可达92％以上．表达蛋白具有良好活性．

Cloning and Expression of Elastase Gene from Pseudomonas sp.

We cloned, expressed and characterized an elastase gene ela from Pseudornonas sp. XZG36, to facilitate the production of elastase using microorganisms. The gene encoded elastase using template of DNA genome from Pseudornonas sp. XZG36 was amplified by PCR and then cloned the open reading frame of ela into pET30a（＋） vector and expressed by Isopropyl β-D-1-thiogalactopyranoside（IPTG） induction. After HisTrap affinity column purification,the characteristics of ELA were studied. We cloned ela gene by PCR and then the gene sequenced as about 1672 bp and encoded polypeptides of 497 amino acid residues, which were consistent with the anticipated length of DNA. The gene was expressed in Escherichia coli BL21（DE3）. The expression product was about 20% of total cell protein,and its relative molecular weight was 48 000, consistent with the anticipated weight analysed by SDS-PAGE. A single strap was clear after recombinant protein was puritied and the purity of recombinant protein was above 92% by SDS-PAGE. The vector containing ela gene was constructed successfully,and ELA recombinant protein was expressed in E. coli BL21（DE3） and showed high activity.