Document Detail


Engineering and refolding of a novel trimeric fusion protein TRAIL-collagen XVIII NC1.
MedLine Citation:
PMID:  23208613     Owner:  NLM     Status:  Publisher    
Abstract/OtherAbstract:
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:
Li Qiang Pan; Zhang Ming Xie; Xiao Jing Tang; Min Wu; Fu Rong Wang; Hua Naranmandura; Shu Qing Chen
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Publication Detail:
Type:  JOURNAL ARTICLE     Date:  2012-12-4
Journal Detail:
Title:  Applied microbiology and biotechnology     Volume:  -     ISSN:  1432-0614     ISO Abbreviation:  Appl. Microbiol. Biotechnol.     Publication Date:  2012 Dec 
Date Detail:
Created Date:  2012-12-4     Completed Date:  -     Revised Date:  -    
Medline Journal Info:
Nlm Unique ID:  8406612     Medline TA:  Appl Microbiol Biotechnol     Country:  -    
Other Details:
Languages:  ENG     Pagination:  -     Citation Subset:  -    
Affiliation:
Department of Pharmacology, Toxicology and Biochemical Pharmaceutics, College of Pharmaceutical Sciences, Zhejiang University, Hangzhou, 310058, China.
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