title: A pH-induced, intein-mediated expression and purification of recombinant human epidermal growth factor in Escherichia coli.
abstract: 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.
authors: Inst. of Microbiology, Shaanxi Province Academy of Sciences, Xi'an, 710043, China.
authors: Yuejuan
authors: Y
authors: Zhang
authors: Inst. of Microbiology, Shaanxi Province Academy of Sciences, Xi'an, 710043, China.
authors: Kun
authors: K
authors: Zhang
authors: Inst. of Microbiology, Shaanxi Province Academy of Sciences, Xi'an, 710043, China.
authors: Yi
authors: Y
authors: Wan
authors: Inst. of Microbiology, Shaanxi Province Academy of Sciences, Xi'an, 710043, China.
authors: Jing
authors: J
authors: Zi
authors: Inst. of Microbiology, Shaanxi Province Academy of Sciences, Xi'an, 710043, China.
authors: Yan
authors: Y
authors: Wang
authors: Inst. of Microbiology, Shaanxi Province Academy of Sciences, Xi'an, 710043, China.
authors: Jun
authors: J
authors: Wang
authors: 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.
authors: Lili
authors: L
authors: Wang
authors: State Key Laboratory of Cancer Biology, Dept. of Biopharmaceutics, School of Pharmacy, Fourth Military Medical University, Xi'an, 710032, China.
authors: Xiaochang
authors: X
authors: Xue
mesh_terms: D002686:Chitin / Q000737:chemistry; D002846:Chromatography, Affinity; D002852:Chromatography, Ion Exchange; D004815:Epidermal Growth Factor / Q000096:biosynthesis* / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D006358:Hot Temperature; D006801:Humans; D006863:Hydrogen-Ion Concentration; D002479:Inclusion Bodies / Q000235:genetics* / Q000378:metabolism; D047668:Inteins; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics
chemical_list: D011993:Recombinant Fusion Proteins; D002686:Chitin; D004815:Epidermal Growth Factor
keywords: heat treatment; intein; ion-exchange chromatography; rhEGF