School of Biological Science and Engineering, Shaanxi University of Technology, Hanzhong, 723001, China; Shaanxi Key Laboratory of Degradable Biomedical Materials, School of Chemical Engineering, Northwest University, Taibai North Road 229, Xi'an, 710069, China.
Department of Gastroenterology and Hepatology, Tianjin Medical University, General Hospital, Tianjin, 300052, China.
Protein Expr Purif. 2020 Sep;173:105634. doi: 10.1016/j.pep.2020.105634. Epub 2020 Apr 21.
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 ELP to ELP was constructed using recursive directional ligation by plasmid reconstruction. ELP was added to the C-terminus of EG12B as a fusion tag to obtain the expression vector pET28-EG12B-ELP, 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-ELP expression level was up to about 35% of the total cellular proteins. After three rounds of ITC, 8.14 mg of EG12B-ELP was obtained from 500-mL lysogeny broth culture medium. The recovery rate and purification fold of EG12B-ELP purified by ITC reached 78.1% and 11.8, respectively. The cellulase activity assay showed that EG12B-ELP had a better thermostability, higher optimal temperature, and longer half-life than those of free EG12B. Overall, our results suggested that ELP could be used as a favorable fusion tag, providing a rapid, simple, and inexpensive strategy for non-chromatographic target-protein purification.
嗜热栖热菌来源的内切葡聚糖酶 EG12B 是一种具有很大工业应用潜力的嗜热纤维素酶。在这里，为了以简单有效的方式实现 EG12B 的选择性纯化，融合了弹性蛋白样多肽（ELP），它作为一种热响应多肽，以实现其反向相转变循环（ITC）。通过质粒重建的递归定向连接构建了一个由 ELP 到 ELP 的小基因文库。将 ELP 添加到 EG12B 的 C 末端作为融合标签，获得表达载体 pET28-EG12B-ELP，将其转化到大肠杆菌 BL21（DE3）中，通过 IPTG 诱导表达融合蛋白。灰度扫描分析表明，EG12B-ELP 的表达水平高达总细胞蛋白的约 35%。经过三轮 ITC，从 500-mL 溶菌肉汤培养基中获得了 8.14mg 的 EG12B-ELP。通过 ITC 纯化的 EG12B-ELP 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-ELP 具有更好的热稳定性、更高的最适温度和更长的半衰期，优于游离 EG12B。总体而言，我们的结果表明，ELP 可以作为一种有利的融合标签，为非色谱靶蛋白的快速、简单、廉价的纯化提供了一种策略。
