Wang Qi, Zhao Jiaojiao, Wang Yan, Sun Honglou, Jiang Yi, Luo Lan, Yin Zhimin
Jiangsu Province Key Laboratory for Molecular and Medicine Biotechnology, College of Life Sciences, Nanjing Normal University, Nanjing, Jiangsu, PR China.
Protein Expr Purif. 2013 Dec;92(2):135-40. doi: 10.1016/j.pep.2013.09.014. Epub 2013 Sep 29.
Human hepassocin (HPS) is a hepatic growth factor which can accelerate hepatocyte proliferation in vivo and protect against liver injury. Previous reports have shown that HPS expressed in Escherichia coli resulted in inclusion bodies or low yield. In this study, the application of small ubiquitin-related modifier (SUMO) fusion technology in combined with four different chaperone teams on the soluble expression of recombinant HPS protein were explored and analyzed. The soluble expression of HPS was improved significantly by SUMO fusion and co-expression with trigger factor (Tf) chaperone, which was identified by SDS-PAGE and Western blotting. The fusion protein was purified to 90% purity by metal chelate chromatography with a yield of 98 mg per liter fermentation culture. Finally, about 19 mg HPS was obtained from 1l of fermentation culture with no less than 96% purity following purification of the SUMO protease cleavage and re-purified by the Ni-NTA resin chromatography, which was the highest yield of HPS reported so far with less time and effort. The recombinant HPS significantly stimulated the proliferation of human hepatic cell line L02 cells. The present work provides an effective system for soluble expression of functional HPS, which will facilitate the clinical developments of recombinant protein drugs.
人肝促生长素（HPS）是一种肝生长因子，可在体内加速肝细胞增殖并预防肝损伤。先前的报道表明，在大肠杆菌中表达的HPS会导致包涵体形成或产量较低。在本研究中，探索并分析了小泛素相关修饰物（SUMO）融合技术与四种不同伴侣蛋白组合对重组HPS蛋白可溶性表达的应用。通过SDS-PAGE和蛋白质免疫印迹法鉴定，SUMO融合以及与触发因子（Tf）伴侣蛋白共表达显著提高了HPS的可溶性表达。通过金属螯合层析将融合蛋白纯化至90%的纯度，每升发酵培养物的产量为98 mg。最后，在经过SUMO蛋白酶切割纯化并通过Ni-NTA树脂层析重新纯化后，从1升发酵培养物中获得了约19 mg纯度不低于96%的HPS，这是迄今为止报道的HPS最高产量，且省时省力。重组HPS显著刺激了人肝细胞系L02细胞的增殖。本研究工作为功能性HPS的可溶性表达提供了一个有效的系统，这将有助于重组蛋白药物的临床开发。
