摘要
目前有关限制性内切酶NotⅠ的性质特征及功能机制等方面的研究日渐增多,但商品化NotⅠ及某些限制性内切酶的价格依然居高不下,其主要原因在于表达量低、提纯程序繁琐、得率低等问题的存在。为探索限制性内切酶NotⅠ提纯的新工艺,从豚鼠耳炎诺卡菌(Nocardia otitidis-caviarum)中克隆出限制性内切酶NotⅠ的基因并使之在大肠杆菌中高效表达。首先将由成团肠杆菌(Enterobacter agglomerans)中克隆所得甲基化酶EagⅠM(EagⅠmethylase gene)基因连接到pBR322载体上,转化大肠杆菌ER2566,将豚鼠耳炎诺卡菌中克隆所得的限制性内切酶NotⅠR(NotⅠrestriction endonuclease gene)基因连接到表达载体pACYC184-PT7上,将此重组质粒转化到上述已转入甲基化重组质粒pBR322-EagⅠM的ER2566中,构建成NotⅠ蛋白表达菌ER2566[pBR322-EagⅠM,pACYC184-PT7-NotⅠR]。重组工程菌经IPTG诱导可表达限制性内切酶NotⅠ,并对诱导条件进行优化使之以可溶形式高效表达。应用KTA purifier 100蛋白纯化系统,对纯化工艺进行创新,通过DEAE Sephrose FF离子交换层析、phenyl HP疏水层析和Superdex 75 10/300GL分子筛层析对蛋白进行提纯。纯化后NotⅠ经酶活力及纯度鉴定,其比活力为1.37×106U/mg,提纯35倍,得率为17.8%,产量达9.8×106 Units/g wet cell,提纯时间缩减为原来的1/10,在产量和效率上较以前报道均有很大提高。该纯化工艺的新方法,为实验室制备及工业化生产Ⅱ型限制性内切酶提供了进一步的借鉴。且该酶的成功获得为后续研究提供了材料,为更多新发现内切酶的成功克隆提供了参考。
In order to study its specificities,the NotⅠR gene was cloned from Nocardia otitidis-caviarum.Firstly,the genome DNA of Enterobacter agglomerans was extracted as template,obtained EagⅠmethylase gene by PCR and connected EagⅠM gene to pBR322 vector to gain recombinant expression plasmid pBR322-Eag ⅠM.Then transformed this plasmid into E.coli 2566.Secondly,extracted the genome DNA from Nocardia otitidis-caviarum as template and obtained the restriction enzyme NotⅠR gene by PCR.After ligating the NotⅠR gene to pACYC184-PT7,the pACYC184-PT7-NotⅠR plasmid was transformed into the ER2566 which was protected through the methylation by pBR322-EagⅠM recombinant plasmid.The engineered strain ER2566 [pBR322-EagⅠM,pACYC184-PT7-NotⅠR] could be induced to express restriction enzymes NotⅠ by IPTG and the induction conditions were optimized to make its expression mostly in soluble form.The enzyme was purified by KTA purifier 100 protein purification system.Through DEAE Sephrose FF,phenyl HP and Superdex 75 10/300 GL molecular sieve chromatography,the Not Ⅰenzyme was purified 35-fold,the yield was 9.8 × 106 Units / g wet cell which was up to17.8% of the crude enzyme and the specific activity of the purified NotⅠwas 1.37 × 106U/mg.Digestion results showed that the enzyme was purified to homogeneity and was free of detectable contamination by other DNase(exo and endo).After optimization of the expression and purification conditions,the yield and efficiency of NotⅠ enzyme were greatly improved in comparison with that previously reported.
出处
《中国生物工程杂志》
CAS
CSCD
北大核心
2011年第8期102-109,共8页
China Biotechnology
基金
黑龙江省博士后科研启动资金
黑龙江省教育厅项目(11521022)资助项目
关键词
限制性内切酶Not
I
克隆
表达
纯化
Not I restriction endonuclease Clone Expression Purification