title: Expression, purification, and characterization of scar tissue neovasculature endothelial cell-targeted rhIL10 in Escherichia coli.
abstract: Interleukin 10 (IL10) plays a pivotal role in the anti-inflammatory response and immunosuppressive reactions. It has also been identified as a new promising therapy for scar formation. Treatment of scars with IL10 has significant effects, but there are some shortcomings, including poor tissue-binding specificity and low effectiveness. RGD peptide has been demonstrated to bind specifically to αvβ3 integrin on neovasculature endothelial cells, and the excess production of neovasculature is crucial to scar formation. To increase efficacy against scar formation and to decrease the side effects on normal tissues, a novel hybrid protein combining human IL10 with RGD was designed. The DNA sequence encoding the recombinant fusion protein IL10-RGD (rhIL10-RGD) was subcloned into a pET22b (+) vector for protein expression in E. coli strain BL21 (DE3). SDS-PAGE analysis displayed an induced expression product band at a molecular weight of 19.3 kDa, which constituted 30 % of the total bacterial protein. We developed a procedure to purify rhIL10-RGD from inclusion bodies and then renatured the protein using dialysis against urea with a step-down concentration procedure. Hypertrophic scar fibroblasts (HSFs) were treated with rhIL10-RGD, and the fibrosis-related protein levels were assessed by Western blotting. The results indicated that rhIL10-RGD can downregulate the expression levels of Col1 and α-SMA in HSFs and suppress tube formation of HUVECs. These results indicate that rhIL10-RGD has anti-fibrosis effects and can potentially be used to treat the neovasculature in scar formation and improve the abnormal deposition of the extracellular matrix (ECM). Thus, rhIL10-RGD may be a more effective candidate for scar-improvement and anti-fibrosis therapy.
authors.affiliation: Department of Burns and Cutaneous Surgery, Xijing Hospital, Fourth Military Medical University, 17 West Changle Road, Xi'an, 710032, China.
authors.forename: Jihong
authors.initials: J
authors.lastname: Shi
authors.forename: Yi
authors.initials: Y
authors.lastname: Wan
authors.forename: Shan
authors.initials: S
authors.lastname: Shi
authors.forename: Jing
authors.initials: J
authors.lastname: Zi
authors.forename: Hao
authors.initials: H
authors.lastname: Guan
authors.forename: Yuejuan
authors.initials: Y
authors.lastname: Zhang
authors.forename: Zhao
authors.initials: Z
authors.lastname: Zheng
authors.forename: Yanhui
authors.initials: Y
authors.lastname: Jia
authors.forename: Xiaozhi
authors.initials: X
authors.lastname: Bai
authors.forename: Weixia
authors.initials: W
authors.lastname: Cai
authors.forename: Linlin
authors.initials: L
authors.lastname: Su
authors.forename: Xiongxiang
authors.initials: X
authors.lastname: Zhu
authors.forename: Dahai
authors.initials: D
authors.lastname: Hu
mesh_terms: D002921:Cicatrix / Q000188:drug therapy / Q000473:pathology; D042783:Endothelial Cells / Q000378:metabolism / Q000473:pathology; D004926:Escherichia coli / Q000235:genetics; D005109:Extracellular Matrix / Q000235:genetics / Q000378:metabolism; D005347:Fibroblasts / Q000378:metabolism; D005355:Fibrosis / Q000188:drug therapy* / Q000473:pathology; D006801:Humans; D016753:Interleukin-10 / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification / Q000627:therapeutic use; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification / Q000627:therapeutic use
chemical_list: C508609:IL10 protein, human; D011994:Recombinant Proteins; D016753:Interleukin-10