Endoglucanase EG12B from Thermotoga maritima is a thermophilic cellulase that has great potential for industrial applications. Here, to enable the selective purification of EG12B in a simple and efficient manner, an elastin-like polypeptide (ELP), which acts as a thermally responsive polypeptide, was fused with EG12B to enable its inverse phase transition cycling (ITC). A small gene library comprising ELPs from ELP5 to ELP50 was constructed using recursive directional ligation by plasmid reconstruction. ELP50 was added to the C-terminus of EG12B as a fusion tag to obtain the expression vector pET28-EG12B-ELP50, which was transformed into Escherichia coli BL21 (DE3) to enable the expression of fusion protein via IPTG induction. Gray scanning analysis revealed that the EG12B-ELP50 expression level was up to about 35% of the total cellular proteins. After three rounds of ITC, 8.14 mg of EG12B-ELP50 was obtained from 500-mL lysogeny broth culture medium. The recovery rate and purification fold of EG12B-ELP50 purified by ITC reached 78.1% and 11.8, respectively. The cellulase activity assay showed that EG12B-ELP50 had a better thermostability, higher optimal temperature, and longer half-life than those of free EG12B. Overall, our results suggested that ELP50 could be used as a favorable fusion tag, providing a rapid, simple, and inexpensive strategy for non-chromatographic target-protein purification.

中文翻译：

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通过与疏水弹性蛋白样多肽融合，从海生热球菌中非稳定纯化内切葡聚糖酶。

滨海热球菌的内切葡聚糖酶EG12B是一种嗜热纤维素酶，在工业应用中具有巨大潜力。在这里，为了能够以简单有效的方式选择性地纯化EG12B，将充当热响应性多肽的弹性蛋白样多肽（ELP）与EG12B融合，使其能够进行逆相变循环（ITC）。使用递归定向连接通过质粒重建来构建包含从ELP5到ELP50的ELP的小基因文库。将ELP50作为融合标签添加到EG12B的C末端，以获得表达载体pET28-EG12B-ELP50，将其转化到大肠杆菌BL21（DE3）中，以通过IPTG诱导表达融合蛋白。灰色扫描分析表明，EG12B-ELP50表达水平高达细胞总蛋白的35％。在三轮ITC之后，从500 mL溶菌性肉汤培养基中获得了8.14 mg EG12B-ELP50。ITC纯化的EG12B-ELP50的回收率和纯化倍数分别达到78.1％和11.8。纤维素酶活性测定表明，EG12B-ELP50具有比游离EG12B更好的热稳定性，更高的最佳温度和更长的半衰期。总体而言，我们的结果表明ELP50可以用作有利的融合标签，为非色谱目标蛋白的纯化提供快速，简单和廉价的策略。与游离EG12B相比，它的最佳温度更高，半衰期更长。总体而言，我们的结果表明ELP50可以用作有利的融合标签，为非色谱目标蛋白的纯化提供快速，简单和廉价的策略。与游离EG12B相比，它的最佳温度更高，半衰期更长。总体而言，我们的结果表明ELP50可以用作有利的融合标签，为非色谱目标蛋白的纯化提供快速，简单和廉价的策略。