Abstract

Step-by-step screening of the main characteristics of refolding buffer is proposed to obtain recombinant antistaphylococcal endolysin LysK containing two catalytic domains, CHAP and amidase-2, in active form from E. coli inclusion bodies. The optimal pH, temperature, redox potential of the refolding buffer, as well as the optimal protein concentration and type of antiaggregation compound were determined. The composition of a renaturation system of antistaphylococcal endolysin containing 20 mM phosphate buffer, pH 7.4, 0.4 M sucrose, and 2.5 mM DTT at 10°C with dilution to a final concentration of ~150 μg/mL was found to be optimal. The refolding yield after scaling was about 29.5 ± 6.7%, which produced 16 ± 2.3 mg of the target protein from 2.25 g of washed inclusion bodies with a specific enzyme activity of 1.8 ± 0.73 × 10³ U/mg.

中文翻译：

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大肠杆菌包涵体活性形式复性条件的筛选及重组抗葡萄球菌溶血素LysK CA的获得

摘要

建议对重折叠缓冲液的主要特征进行分步筛选，以从大肠杆菌中获得以活性形式包含两个催化结构域CHAP和酰胺酶-2的重组抗葡萄球菌溶血素LysK 。包涵体。确定了最佳pH，温度，重折叠缓冲液的氧化还原电势以及最佳蛋白质浓度和抗聚集化合物的类型。发现在10°C下稀释至最终浓度约为150μg/ mL的抗葡萄球菌溶血素的复性系统的组成是最佳的，该系统包含20 mM磷酸盐缓冲液，pH 7.4、0.4 M蔗糖和2.5 mM DTT。结垢后的重折叠产率约为29.5±6.7％，由2.25 g洗涤过的包涵体产生16±2.3 mg的目标蛋白，比酶活性为1.8±0.73×10 ³ U / mg。