Zhang Qing, Zhao Xiaozhi, Xu Xiaoyu, Tang Bo, Zha Zhenglei, Zhang Mingxin, Yao Dongwei, Chen Xiaoxiang, Wu Xuhong, Cao Lin, Guo Hongqian
Department of Urology, Drum Tower Hospital, Medical School of Nanjing University, 321 Zhongshan Road, Nanjing 210008, Jiangsu, PR China.
Vazyme Biotech Co., Ltd, Nanjing 210000, Jiangsu, PR China.
Protein Expr Purif. 2014 Dec;104:14-9. doi: 10.1016/j.pep.2014.09.010. Epub 2014 Sep 26.
Human cystatin C (CYSC) is a 13-kDa endogenous cysteine proteinase inhibitor and was investigated as a replacement for creatinine as a marker of renal function. However, expressing recombinant CYSC is difficult in Escherichia coli because of resulting low yield and insufficient purity and insolubility. Here, we cloned and fused CYSC to the C-terminus of three soluble partners - maltose-binding protein (MBP), glutathione S-transferase (GST) and translation initiation factor 2 domain I (IF2) - to screen for their ability to improve the solubility of recombinant CYSC when expressed in E. coli. MBP was best at enhancing the soluble expression of CYSC, with soluble fractions accounting for 92.8±3.11% of all proteins. For scaled production, we purified the de-tagged CYSC by using a 3C protease-cleaved MBP-T3-CYSC fused protein with immobilized metal affinity chromatography and cation-affinity purification. The molecular weights of the de-tagged CYSC and human natural CYSC were similar, and the former could react specifically with CYSC polyclonal antibody. Moreover, the de-tagged CYSC displayed full biological activity against papain and cathepsin B, which was very similar to that of the human natural CYSC protein standard. We provide a method to produce large amounts of soluble recombinant human CYSC in E. coli.
人胱抑素C（CYSC）是一种13 kDa的内源性半胱氨酸蛋白酶抑制剂，作为肾功能标志物，它被研究用作肌酐的替代物。然而，在大肠杆菌中表达重组CYSC很困难，因为产量低、纯度不足且不溶性高。在此，我们将CYSC克隆并融合到三种可溶性伴侣蛋白——麦芽糖结合蛋白（MBP）、谷胱甘肽S-转移酶（GST）和翻译起始因子2结构域I（IF2）——的C末端，以筛选它们在大肠杆菌中表达时提高重组CYSC溶解度的能力。MBP在增强CYSC的可溶性表达方面效果最佳，可溶性组分占所有蛋白质的92.8±3.11%。为了进行规模化生产，我们使用固定化金属亲和色谱和阳离子亲和纯化，通过3C蛋白酶切割的MBP-T3-CYSC融合蛋白纯化去除标签的CYSC。去除标签的CYSC和人天然CYSC的分子量相似，前者能与CYSC多克隆抗体发生特异性反应。此外，去除标签的CYSC对木瓜蛋白酶和组织蛋白酶B表现出完全的生物学活性，这与人类天然CYSC蛋白标准品非常相似。我们提供了一种在大肠杆菌中大量生产可溶性重组人CYSC的方法。
