title: Expression and purification of rhIL-10-RGD from Escherichia coli as a potential wound healing agent.
abstract: Various protocols for recombinant Interleukin-10 (IL-10) purification in wound healing have been reported previously. However, the therapeutic effect was not obvious. Thus, it is of great importance to find new and effective approaches for therapy. In this study, we propose that IL-10 and Arginine-Glycine-Aspartic (RGD) peptide would be a valuable therapeutic for wound healing. To explore a high-efficiency and cost-effective approach for the production of IL-10 and RGD peptide with bioactivity, a synthetic gene was cloned into a recombinant pTWIN1 vector. As a consequence, rhIL-10-RGD and the pH-induced self-cleavable Ssp DnaB mini-intein as a fusion protein was highly expressed by IPTG induction in Escherichia coli Rosetta without extra residues in a bioreactor. After Ni affinity chromatographic purification, rhIL-10-RGD was released by the Ssp DnaB intein-mediated self-cleavage that is triggered by pH shift. SDS-PAGE and silver staining showed a major band with an estimated molecular mass of 19.3kDa. Cell proliferation assay confirmed its potent proliferation activity on MC/9 murine mast cells. In conclusion, we report a novel strategy to produce rhIL-10-RGD mediated by the pH-induced self-cleavable Ssp DnaB mini-intein, and show that rhIL-10-RGD could play an effective role in wound healing of BALB/c mice.
authors.affiliation: Department of Burns and Cutaneous Surgery, Xijing Hospital, Fourth Military Medical University, 127 West Chang-le Road, Xi'an 710032, China. Electronic address: cy1212@outlook.com.
authors.forename: Fangfang
authors.initials: F
authors.lastname: Yang
authors.affiliation: Shaanxi Microbiology Institute, Shaanxi Academy of Science, 76 Xiying Road, Xi'an 710043, China.
authors.forename: Yi
authors.initials: Y
authors.lastname: Wan
authors.affiliation: Department of Burns and Cutaneous Surgery, Xijing Hospital, Fourth Military Medical University, 127 West Chang-le Road, Xi'an 710032, China.
authors.forename: Jiaqi
authors.initials: J
authors.lastname: Liu
authors.affiliation: Department of Burns and Cutaneous Surgery, Xijing Hospital, Fourth Military Medical University, 127 West Chang-le Road, Xi'an 710032, China.
authors.forename: Xuekang
authors.initials: X
authors.lastname: Yang
authors.affiliation: Department of Burns and Cutaneous Surgery, Xijing Hospital, Fourth Military Medical University, 127 West Chang-le Road, Xi'an 710032, China.
authors.forename: Hongtao
authors.initials: H
authors.lastname: Wang
authors.affiliation: Department of Burns and Cutaneous Surgery, Xijing Hospital, Fourth Military Medical University, 127 West Chang-le Road, Xi'an 710032, China.
authors.forename: Ke
authors.initials: K
authors.lastname: Tao
authors.affiliation: Department of Burns and Cutaneous Surgery, Xijing Hospital, Fourth Military Medical University, 127 West Chang-le Road, Xi'an 710032, China.
authors.forename: Juntao
authors.initials: J
authors.lastname: Han
authors.affiliation: Department of Burns and Cutaneous Surgery, Xijing Hospital, Fourth Military Medical University, 127 West Chang-le Road, Xi'an 710032, China.
authors.forename: Jihong
authors.initials: J
authors.lastname: Shi
authors.affiliation: Department of Burns and Cutaneous Surgery, Xijing Hospital, Fourth Military Medical University, 127 West Chang-le Road, Xi'an 710032, China. Electronic address: hudhai@fmmu.edu.cn.
authors.forename: Dahai
authors.initials: D
authors.lastname: Hu
mesh_terms: D000818:Animals; D002846:Chromatography, Affinity; D003001:Cloning, Molecular; D055808:Drug Discovery; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D005822:Genetic Vectors; D016753:Interleukin-10 / Q000235:genetics / Q000302:isolation & purification* / Q000627:therapeutic use*; D051379:Mice; D008807:Mice, Inbred BALB C; D009842:Oligopeptides / Q000235:genetics / Q000302:isolation & purification* / Q000627:therapeutic use*; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification / Q000378:metabolism / Q000627:therapeutic use; D014945:Wound Healing; D014947:Wounds and Injuries / Q000628:therapy
chemical_list: C508609:IL10 protein, human; D009842:Oligopeptides; D011993:Recombinant Fusion Proteins; D016753:Interleukin-10; C047981:arginyl-glycyl-aspartic acid
keywords: Fusion protein; Purification; Wound healing; rhIL-10-RGD