拟南芥阿拉伯糖-5-磷酸异构酶的原核表达、纯化及酶催化特性

Expression, purification and characterization of arabinose-5-phosphate isomerase from Arabidopsis thaliana

摘要:

阿拉伯糖-5-磷酸异构酶 (KdsD) 是2-酮-3-脱氧辛糖酸 (KDO) 生物合成途径的第一个关键限速酶，通过无缝克隆技术将拟南芥KdsD基因构建至原核表达载体pET-HTT，经过IPTG诱导，在大肠杆菌BL21 (DE3)中获得了大量重组蛋白的可溶性表达；表达产物经Ni-NTA亲和层析和分子筛层析 (SEC) 方法进行酶蛋白的分离纯化步骤，得到纯度85%以上的高纯度酶；分子筛层析结果发现纯化后的目的蛋白KdsD在溶液中主要以多聚体、二聚体和单体形式存在，这同微生物来源KdsD酶在溶液中以四聚体形式存在很大差异；进一步使用Western blotting和MALDI-TOF MASS技术对纯化的蛋白进行鉴定；测定了拟南芥KdsD酶学性质，证明该酶催化反应的最适pH值为8.0，最适作用温度为37 ℃，各种金属离子在低浓度均对酶活性存在不同程度的抑制作用，其中以Co2+、Cd2+对酶活性的抑制作用最强，而5 mmol/L金属螯合剂EDTA对酶有激活作用。此外，以阿拉伯糖-5-磷酸 (A5P) 为底物时，拟南芥KdsD酶动力学常数Vmax和Km值分别为0.18 mmol/(L·min)、0.16 mmol/L，比较发现该酶与底物的亲和性高于大肠杆菌KdsD。以上研究结果为KdsD蛋白结构与功能及其在新型抗生素研制领域中的工业化应用奠定了基础。

Abstract:

Arabinose-5-phosphate isomerase (KdsD) is the first key limiting enzyme in the biosynthesis of 3-deoxy-D-manno-octulosonate (KDO). KdsD gene was cloned into prokaryotic expression vector pET-HTT by seamless DNA cloning method and the amount of soluble recombinant protein was expressed in a soluble form in E. coli BL21?(DE3) after induction of Isopropyl β-D-1-thiogalactopyranoside (IPTG). The target protein was separated and purified by Ni-NTA affinity chromatography and size exclusion chromatography, and its purity was more than 85%. Size exclusion chromatography showed that KdsD protein existed in three forms: polymers, dimmers, and monomers in water solution, different from microbial KdsD enzyme with the four polymers in water solution. Further, the purified protein was identified through Western blotting and MALDI-TOF MASS technology. The results of activity assay showed that the optimum pH and temperature of AtKdsD isomerase activities were 8.0 and 37 ℃, respectively. The enzyme was activated by metal protease inhibitor EDTA (5 mmol/L) and inhibited by some metal ions at lower concentration, especially with Co2+ and Cd2+ metal ion. Furthermore, when D-arabinose-5-phosphate (A5P) was used as substrate, Km and Vmax of AtKdsD values were 0.16 mmol/L, 0.18 mmol/L·min. The affinity of AtKdsD was higher than KdsD in E. coli combined with substrate. Above results have laid a foundation for the KdsD protein structure and function for its potential industrial application.