芽孢杆菌弹性蛋白酶基因的克隆、表达及其大豆肽的制备能力鉴定
Cloning and expression of the elastase gene Bacillus subtilis and identification of the ability of preparation of soybean peptide
-
摘要: 从芽孢杆菌中克隆得到弹性蛋白酶并命名为TX1,并连入p EAZY E1载体,构建了原核表达载体p EAZY E1-TX1。重组质粒导入大肠杆菌BL21后,经IPTG诱导基因表达。表达产物经Ni柱纯化后通过p H-stat测定其制备大豆肽的水解能力。序列分析结果表明TX1基因长度为1143bp,与Genbank上已报道序列(JQ305692.1)同源性最高为99.48%,产生的碱基突变不影响其氨基酸编码。蛋白表达分析结果表明,当IPTG浓度为0.3mmol·L-1,诱导4h蛋白表达量最高。经SDS-PAGE和Western分析结果表明,经Ni离子亲和层析纯化获得量大小为39.4ku的弹性蛋白酶His-TX1。初步确定其在50℃、p H为7.4反应4h时的大豆肽水解率最高,达到14.51%。Abstract: A elastase gene named TX1 was isolated from the genomic DNA of Bacillus subtilis by PCR amplification, and constructed into p EAZY E1 vector to generate a recombinant expression plasmid p EAZY E1-TX1. The recombinant plasmid was transformed into E.coli BL21. The expression was induced by IPTG and the fusion protein His-TX1 was purified by Ni-NTA. The ability of the purified expression of hydrolysis of Soybean Peptide was identified by p H-stat method. The sequence analysis suggested that TX1 gene was cloned with a length of 1143 bp, had 99.48% homology with the Elastase gene (JQ305692.1) published in the Gen Bank, and the mutants would not infected the sequence of the amimo acids encoded. The analysis of expression showed that the highest expression level was obtained with 0.3mmol·L-1IPTG at 28℃ for 4h. The SDS-PAGE and Western blotting analysis showed that fusion protein which was 39.4ku purified with nickel-nitrilotriacetic acid (Ni-NTA) metal-affinity chromatography matrices was the Elastase His-TX1. The highest hydrolysis activity of HIS-TX1 was detected at 50℃, p H7.4 for 4h, the hydrolysis reached 14.51%.