title: Engineering and refolding of a novel trimeric fusion protein TRAIL-collagen XVIII NC1.
abstract: Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is considered to be a promising anticancer agent because its active form TRAIL trimer is able to induce apoptosis in different tumor cell lines while sparing normal cells. However, TRAIL trimer possesses a short half-life and low stability, which turns out to be a major obstacle for the development of clinical trials. In our present study, we constructed a recombined TRAIL trimer by genetic fusion of non-collagenous domain (NC1) of human collagen XVIII or its trimerization domain (TD) to C-terminus of TRAIL via a flexible linker, and then refolded the fusion proteins using a two-step refolding approach, namely a combination of dilution and gel filtration chromatography. As a result, both recombinant proteins, TRAIL-NC1 and TRAIL-TD, were expressed in Escherichia coli as inclusion bodies, and they exhibited difficultly to refold efficiently by conventional methods. Thereby, we applied a modified two-step refolding approach to refold fusion proteins. More than 55 % of TRAIL-NC1 and 90 % of TRAIL-TD protein activity was recovered during the two-step refolding approach, and their stability was also increased significantly. Also, size exclusion chromatography showed refolded TRAIL-NC1 was a trimer while TRAIL-TD, hexamer. However, both of them exerted good apoptosis activity on NCI-H460 cells.
authors.affiliation: Department of Pharmacology, Toxicology and Biochemical Pharmaceutics, College of Pharmaceutical Sciences, Zhejiang University, Hangzhou 310058, China.
authors.forename: Li Qiang
authors.initials: LQ
authors.lastname: Pan
authors.forename: Zhang Ming
authors.initials: ZM
authors.lastname: Xie
authors.forename: Xiao Jing
authors.initials: XJ
authors.lastname: Tang
authors.forename: Min
authors.initials: M
authors.lastname: Wu
authors.forename: Fu Rong
authors.initials: FR
authors.lastname: Wang
authors.forename: Hua
authors.initials: H
authors.lastname: Naranmandura
authors.forename: Shu Qing
authors.initials: SQ
authors.lastname: Chen
mesh_terms: D000970:Antineoplastic Agents / Q000302:isolation & purification / Q000378:metabolism / Q000494:pharmacology; D017209:Apoptosis; D045744:Cell Line, Tumor; D002850:Chromatography, Gel; D043170:Collagen Type XVIII / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism* / Q000494:pharmacology; D004847:Epithelial Cells / Q000187:drug effects / Q000502:physiology; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D006801:Humans; D017510:Protein Folding; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism / Q000494:pharmacology; D053221:TNF-Related Apoptosis-Inducing Ligand / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism* / Q000494:pharmacology
chemical_list: D000970:Antineoplastic Agents; D043170:Collagen Type XVIII; D011993:Recombinant Fusion Proteins; D053221:TNF-Related Apoptosis-Inducing Ligand; C505653:TNFSF10 protein, human