【作者】 吕宁；

【导师】 陈鹏；

【作者基本信息】 西北农林科技大学 ， 生物化学与分子生物学， 2016， 硕士

【摘要】 核酸污染是蛋白质重组表达及制备过程存在的一项重大问题。传统的物理、化学方法去除核酸成本较高且操作复杂,酶法去除蛋白质样品中的核酸具有高效便捷的显著优势。灵杆菌核酸酶由于具有高活性、高稳定性等特点而被广泛应用,但如何高效去除添加的核酸酶成为新的生产问题。本研究基于Strep tag II与链霉亲和素突变体之间强的亲和力,构建融合Strep tag II的灵杆菌核酸酶(Strep tag II-S.marcescens nuclease,SNU)和链霉亲和素突变体的重组表达系统,利用固定化链霉亲和素突变体建立灵杆菌核酸酶的高效去除系统。研究取得如下结果:根据灵杆菌核酸酶的基因序列设计融合Strep tag II编码序列的引物,克隆目标基因,构建融合表达载体pLLP-Omp A+snu,并转化宿主菌BL21进行诱导表达。融合Strep tag II的核酸酶以包涵体形式过量表达。在优化高纯度包涵体纯化条件基础上,通过梯度透析可获得高活性的融合蛋白,复性率接近60%。复性蛋白经DEAE-Sepharose CL 6B阴离子交换层析进一步纯化获得了高纯度的融合蛋白。融合蛋白的纯化效率可以达到18.695 mg/L。纯化的融合Strep tag II的灵杆菌核酸酶活性检测试验表明该融合蛋白能够高效降解DNA与RNA,其最适温度为37℃,最适pH为8.0,比活力达到1.53×105 U/mg。低于500 mmol/L的尿素对酶活性几乎没有影响,而高于50 mmol/L MgCl2、EDTA则会对酶活性产生抑制作用。不同二价金属离子对酶活性影响的实验结果表明,Mg2+和Mn2+对重组核酸酶活性有明显的促进作用,Zn2+、Cu2+和Ca2+则没有促进作用,而Co2+、Ni2+和Fe2+对重组核酸酶活性的促进作用不显著。设计引物扩增链霉亲和素序列,并构建原核表达载体p ET28a-St。通过Quikchange点突变技术对链霉亲和素的三个氨基酸位点进行点突变,分别为E44V、S45T、V47R,突变载体命名为pET28a-StM。转化大肠杆菌BL21(DE3)进行诱导表达,目标蛋白以包涵体形式表达,通过包涵体纯化、溶解、复性获得与Strep tag II高亲和的链霉亲和素突变体蛋白。以1,4-丁二醇双缩水甘油醚环氧基介导的蛋白偶联技术将链霉亲和素突变体固定化于Sepharose CL 6B上,并检测链霉亲和素突变体层析柱去除融合Strep tag II的灵杆菌核酸酶的效果。结果表明3 mL的链霉亲和素突变体-Sepharose CL 6B能够将10μg重组核酸酶彻底去除。最终完成了基于链霉亲和素突变体与Strep tag II标签之间高亲和力的灵杆菌核酸酶去除系统的建立,为灵杆菌核酸酶的实践应用奠定了基础。更多还原

【Abstract】 Nucleic acid contamination has become a big problem during the process of expression and preparation of recombinant protein. Traditional methods for removal of nucleic acid are high cost and complicated while nuclease shows high efficiency and convenience. S. marcescens nucleases are widely used due to its high activity and strong stability. However, the arising problem is how to efficiently remove the trace amount nuclease left in the reaction system. In the research, based on the high affinity between Strep tag II and streptavidin, the systems for expressing S. marcescens nucleases fused with Strep tag II and streptavidin were established and the highly efficient removal system was established based on the immobili zed streptavidin mutant. The results are as follow:The primers were designed based on the sequence of S. marcescens nuclease published in GenBank and the sequence of Strep tag II was fused into the forward primer. The nuclease gene was amplified by PCR and ligated into expression vector pLLP-OmpA. The verified recombinant vector pLLP-OmpA+snu was transformed into Escherichia coli BL21 and induced by IPTG, and the recombinant nuclease was overexpressed in the form of inclusion body. The purified inclusion body was dissolved in 8 M urea and renatured by gradient dialysis. The ratio of purity of renatured protein from inclusion body was approximate 60%. The renatured nuclease was further purified through DEAE- Sepharose and the final yield of the fusion protein was 18.695 mg/L.Catalytic characteristics analysis results of S. marcescens fused with Strep tag II showed optimal temperature of recombinant nuclease was 37 ℃ and optimal pH was 8.0 and its specific activity was 1.53 ×105 U/mg. The catalytic activity was not inhibited by urea of 500 mmol/L while was inhibited by EDTA. Mg2+ was necessary for the catalytic activity of the nuclease while inhibited by over 50 mmol/L MgCl2. Evaluation on the effects of differe nt bivalent metal ic cations on catalytic activities showed that the activity was strongly enhanced by Mn2+, Mg2+ and lightly enhanced by Co2+, Ni2+ and Fe2+ while not by Zn2+, Cu2+ and Ca2+.The streptavidin gene was synthetized and ligated into expression vector pET-28 a. By Quikchange site-directed mutagenesis three amino acids were mutated and the three mutants separately were E44V、S45T、V47R. The mutational recombinant vector was named pET28 aStM which was transformed into Escherichia coli BL21(DE3). The streptavidin mutant was overexpressed in the form of inclusion body. The purified streptavidin mutant was obtained by the purification, dissolution, renaturation. The streptavidin mutant was then coupled to Sepharose CL 6B and the effect of removal system was verified with the streptavidin mutant chromatographic column and S. marcescens nuclease fused with Strep tag II. The result showed that 10 μg recombinant nuclease were completely removed by 3 mL streptavidin mutantSepharose CL 6B. Finally the removal system for S. marcescens nuclease has been completed and provides the foundation for the practice application of S. marcescens nuclease.更多还原