摘要
商品化的乙醛脱氢酶主要从动物细胞、肝细胞线粒体中提取,其来源受到很大的限制,而且价格昂贵。为了解决乙醛脱氢酶原料有限的问题,利用微生物发酵法获取乙醛脱氢酶。将人乙醛脱氢酶2基因(aldh2)克隆至原核表达载体p ET32a中,构建重组载体p ET32a-ALDH2。将构建的重组载体转化大肠杆菌E.coli BL21(DE3),用异丙基硫代-β-D-半乳糖苷(IPTG)进行诱导,SDS-PAGE电泳结果显示目的蛋白得到大量表达;且对诱导表达条件进行优化。结果显示,在37℃,1.0 mmol/L IPTG诱导表达6 h为最优表达条件。由于目的蛋白几乎都是以包涵体形式表达,为了得到有活性的乙醛脱氢酶,对包涵体变性、复性和纯化进行了研究。试验数据显示,酶的最终得率为14.49 U/L。并通过用Bradford法,测定复性蛋白的浓度约为77μg/m L,进行活性检测表明,该复性蛋白具有乙醛脱氢酶活性,酶活性约为1.449 U/m L。通过构建重组载体p ET32a-ALDH2和优化表达条件,aldh2在原核表达宿主E.coli BL21(DE3)得到大量表达;此外,利用透析复性法得到的复性蛋白酶活性有所提高。
The commercial acetaldehyde dehydrogenase is mainly extracted from animal cell and hepatocellular mitochondria, Source is limited and the cost is high. So the micobiological fermentation was used to obtain large amounts of aldh2, which had been cloned into the expression vector pET32a with 6His tag that was advantageous to purify the recombinant protein by nickel-chelate chromatography, then the recombinant was transformed into E.coli BL21 ( DE3 ) . After induced by IPTG, the recombinant protein had been expressed and their molecular masses were determined to be approximately 55 kD by SDS-PACE. Through the optimization of inducing expression conditions, the results showed that the condition ( 37℃, 1.0 mmol/L IPTG, induced for 6 h ) is the optimal. Because all target protein were almost expressed in the form of inclusion body, the bioaetive acetaldehyde dehydrogenase was obtained by means of degeneration, purification and renaturation for the inclusion body, and measured the concentration of the renaturated protein by the Bradford method that was 77 μg/mL. Experimental data showed that the final yield of enzyme was 14.49 U/L. Activity tests showed that the protein has the acetaldehyde dehydrogenase activity of about 1.449 U/ mL. By constructing a recombinant vector pET32a-ALDH2 and optimizing the expression condition, aldh2 in prokaryotic expression host get a lot of expression. In addition, the activity of renaturation protein was improved by the dialysis renaturation method.
出处
《生物技术通报》
CAS
CSCD
北大核心
2014年第12期201-206,共6页
Biotechnology Bulletin
基金
国家自然科学基金项目(81172178)