Ramkumar Subramani, Rabindranath Pai Vaishnavo, Thangadurai Chinnathambi, Priya Murugan Vidhya
Genetic Engineering Unit, Centre for Biotechnology, Anna University, Sardar Patel Road, Guindy, Chennai 600 025, India.
J Genet Eng Biotechnol. 2017 Jun;15(1):179-185. doi: 10.1016/j.jgeb.2016.12.006. Epub 2017 Jan 13.
High throughput expression of proteins is often hampered by the failure of certain proteins to express in the particular host strain used for the study. The identification of a host strain compatible for a wide variety of proteins is desirable. In this study, the recombinant expression of therapeutic proteins Erythropoietin (EPO), Streptokinase (SK) and Tumor Necrosis Factor Receptor Extra cellular domain (TNFR ED) that vary widely in their chemical nature was studied in four different strains of namely BL21 (DE3), BL21 (DE3) pLys S, BL21 (DE3) Rosetta pLys S and GJ1158. Since there are no previous report for the analysis of expression and solubility of the above mentioned proteins we studied the same in various stains. Here we report that strain GJ1158 which uses salt induction was found to be the most suitable for overexpression of all the three proteins. Interestingly rare codons were found not to play any significant role in the expression. Protein toxicity and aggregation propensity were also studied. One of the major factors influencing expression was the tendency of the protein to aggregate which in turn influences folding and toxicity levels. The solubility of the proteins was inversely proportional to aggregation. Expression levels were in the order of TNFR ED < EPO < SK. In conclusion, it was observed that GJ1158, a strain known to decrease aggregation of proteins was found to be more suited for expression. This is the first time GJ1158 has been included in this kind of analysis for comparison of protein expression in various hosts.
某些蛋白质在用于研究的特定宿主菌株中无法表达，这常常阻碍了蛋白质的高通量表达。因此，需要鉴定一种能兼容多种蛋白质的宿主菌株。在本研究中，我们在四种不同的菌株，即BL21(DE3)、BL21(DE3)pLys S、BL21(DE3)Rosetta pLys S和GJ1158中，研究了化学性质差异很大的治疗性蛋白质促红细胞生成素(EPO)、链激酶(SK)和肿瘤坏死因子受体胞外域(TNFR ED)的重组表达。由于此前没有关于上述蛋白质表达和溶解性分析的报道，我们在各种菌株中对其进行了研究。在此我们报告，发现使用盐诱导的GJ1158菌株最适合这三种蛋白质的过表达。有趣的是，稀有密码子在表达中并未发挥任何显著作用。我们还研究了蛋白质毒性和聚集倾向。影响表达的一个主要因素是蛋白质聚集的倾向，这反过来又影响折叠和毒性水平。蛋白质的溶解度与聚集呈反比。表达水平顺序为TNFR ED < EPO < SK。总之，我们观察到，已知能减少蛋白质聚集的GJ1158菌株更适合表达。这是GJ1158首次被纳入此类分析，用于比较不同宿主中的蛋白质表达。
