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

摘要:

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0.5mol/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30min，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。

Abstract:

Abstract:［Objective］This paper reports the purification of xylanase using the shortest，sensitive ELP [KV8 F-20]［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．［Results］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］Elastinlike 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.