以类弹性蛋白多肽为标签的表达质粒构建及其用于木聚糖酶的非色谱纯化  

Construction of an expression vector with elastin-like polypeptide tag to purify xylanase

作　　者：付晓平[1] 王文研[1] 张光亚[1] 

机构地区：[1]华侨大学化工学院生物工程与技术系,厦门361021

出　　处：《微生物学报》2012年第1期90-95,共6页Acta Microbiologica Sinica

基　　金：国家自然科学基金(20806031);福建省自然科学基金(2007J0360);华侨大学基本科研业务费专项基金(JB-GJ1006);福建省高校新世纪优秀人才支持计划(07176C02)~~

摘　　要：【目的】旨在构建一个能以非色谱纯化目标蛋白的表达质粒,使用自行设计的类弹性蛋白多肽(ELPs)作为非色谱纯化标签,以纯化目标蛋白。该ELPs长度短,对盐非常敏感。【方法】从头设计了木聚糖酶,将其通过一段无规则卷曲同ELPs相连,合成了编码上述序列的基因,并构建重组表达载体pET-22b-SoxB-M2-S-ELP,转化至大肠杆菌BLR(DE3)中诱导表达,采用可逆相变循环经高速离心纯化木聚糖酶,并考察纯酶的酶学性质。【结果】成功构建了表达载体并表达,在pH=7.0时0.5 mol/L碳酸钠可使ELPs的相变温度降至22℃。在上述条件下,对木聚糖酶进行了非色谱纯化,其纯化倍数为3.2,回收率为21.2%,纯度为64.3%。经测定,未连接ELPs的酶、粗酶及纯化酶学性质基本一致,其最适温度为60℃,最适pH为6.0,最适反应时间为30 min,粗酶70℃保温1 h相对酶活仍有50%,为嗜热木聚糖酶,与预期相符。【结论】ELPs作为非色谱纯化标签纯化重组木聚糖酶具有操作简单、易于放大、成本较低的优势,故所构建的重组质粒可望通用于分离多种重组蛋白,具有较广泛的用途。[Objective] This paper reports the purification of xylanase using the shortest, sensitive ELP ~ KVsF-20J. [Methods] We designed a thermophilic xylanase gene, and recombined it with the ELP via a random coil sequence to generate the vector pET-22b-SoxB-M2-S-ELP. The expressed xylanase was purified by inverse transition cycling through high-speed centrifugation, and then we characterized the purified xylanase. [ Resultsl The phase transition temperature of the ELPs dropped to 22℃ with 0. 5 mol/L sodium carbonate （pH =7）. Under this condition, SoxB-M2-S-ELP was purified by 3. 16 folds after centrifugation. The recovery rate was 21.2% , and purity of the xylanase was 64. 3%. [ Conclusion] Elastin- like polypeptide as a purification tag to purify recombinant proteins is simple, fast, gentle and cheaper. The expression vector we constructed here might be a very useful and reliable tool to purify many other target proteins.

关 键 词：类弹性蛋白多肽 非色谱纯化 相变温度 木聚糖酶 重组质粒 从头设计 

分 类 号：Q814[生物学—生物工程]