摘要
目的建立高产量和高活力的地衣芽胞杆菌碱性蛋白酶基因表达体系。方法采用PCR技术克隆获得目的基因,将其连入表达质粒pET-32 a构建原核表达重组质粒,经测序鉴定后,转化BL21大肠埃希菌,不同温度下IPTG诱导表达融合蛋白,测定酶活;进一步对该基因和编码蛋白进行同源性比较和酶学性质分析。结果碱性蛋白酶基因序列全长1 149 bp,编码382个氨基酸,同源性为99%,融合蛋白分子质量为62 kD,蛋白酶酶活为29 000 U/mL,并且在25℃时是以可溶蛋白形式表达,37℃时部分蛋白以包涵体形式存在。结论此种表达体系可以成功表达具有生物活性的碱性蛋白酶,诱导温度对蛋白酶存在形式具有较大影响。
Objective To establish the expression system of high yield and high activity of Bacillus licheniformis alkaline protease gene.Method Bacillus licheniformis alkaline protease gene was amplified by PCR and ligated to the vector pET-32a.After identification by sequencing,the recombinant protein was expressed in E.coli BL21 though IPTG in different temperature and enzyme activity was determined.The homology of this gene and code protein was compared,enzymology characteristics of which were analyzed.Result The size of amplified gene was 1 149 bp with 382 amino acids coded,and the consistency of sequencing was 99%.The protein molecular weight was 62 kD and the determined enzyme activity was 29 000 U/mL.Alkaline protease was expressed as soluble protein at 25 ℃ and the part of the protein existed as inclusion body at 37 ℃.Conclusion The alkaline protease with biological activity was successfully expressed,and the induction temperature has a greater impact on existing forms of the enzyme.
出处
《中国微生态学杂志》
CAS
CSCD
2011年第7期597-599,604,共4页
Chinese Journal of Microecology
基金
安徽省教育厅自然科学基金项目(KJ2011A107)
长三角联合攻关项目(10140702021)资助
关键词
地衣芽胞杆菌
碱性蛋白酶基因
克隆
表达特性
Bacillus licheniformis； Alkaline protease gene； Cloning； Expression properties；
