Zhang Yuejuan, Zhang Kun, Wan Yi, Zi Jing, Wang Yan, Wang Jun, Wang Lili, Xue Xiaochang
Inst. of Microbiology, Shaanxi Province Academy of Sciences, Xi'an, 710043, China.
Key Laboratory of Synthetic and Natural Functional Molecule Chemistry of Ministry of Education, Shaanxi Key Laboratory of Modern Separation Science, Institute of Modern Separation Science, Northwest University, Xi'an, 710069, China.
Biotechnol Prog. 2015 May-Jun;31(3):758-64. doi: 10.1002/btpr.2086. Epub 2015 Apr 10.
Human epidermal growth factor (hEGF) is a cellular factor that promotes cell proliferation and has been widely used for the treatment of wounds, corneal injuries, and gastric ulcers. Recombinant hEGF (rhEGF) has previously been expressed using the pTWIN1 system with pH-induced intein and a chitin-binding domain. The rhEGF protein can be purified by chitin affinity chromatography because of the high affinity between the chitin-binding domain fusion-tag and the column. However, uncontrolled cleavage presents a major problem with this method. To overcome this problem, a novel purification method has been developed for a pH-induced intein tag rhEGF that is expressed in Escherichia coli. Following purification by denaturation of inclusion bodies, the fusion protein is renatured and simultaneously induced to self-cleave by dialysis. Further purification of rhEGF is achieved by heat treatment and ion-exchange chromatography. Our results show that the purity of rhEGF obtained through this method is over 98% and the quantity of purified rhEGF is 248 mg from a 1 L culture or 2,967 mg from a 12 L culture. Therefore, we conclude that we have developed an efficient purification method of rhEGF, which may be used for the purification of other heat-resistant and acid-resistant recombinant proteins.
人表皮生长因子（hEGF）是一种促进细胞增殖的细胞因子，已广泛用于治疗伤口、角膜损伤和胃溃疡。重组人表皮生长因子（rhEGF）此前已通过带有pH诱导内含肽和几丁质结合域的pTWIN1系统进行表达。由于几丁质结合域融合标签与柱之间的高亲和力，rhEGF蛋白可通过几丁质亲和色谱法进行纯化。然而，不受控制的切割是该方法的一个主要问题。为克服这一问题，已开发出一种用于在大肠杆菌中表达的pH诱导内含肽标签rhEGF的新型纯化方法。通过包涵体变性进行纯化后，融合蛋白复性并同时通过透析诱导自切割。通过热处理和离子交换色谱法进一步纯化rhEGF。我们的结果表明，通过该方法获得的rhEGF纯度超过98%，从1 L培养物中纯化得到的rhEGF量为248 mg，从12 L培养物中纯化得到的rhEGF量为2967 mg。因此，我们得出结论，我们已开发出一种高效的rhEGF纯化方法，该方法可用于纯化其他耐热和耐酸的重组蛋白。
