Screening of Peptide Inhibitors of TACE from a Phage Display Random 15-Peptide Library by Recombinant TACE Ectodomain  

作　　者：Huang Wei Yang Yuzhen Wang Zhen Hang Ling 

机构地区：[1]Department of Biochemistry and Molecular Biology,Tongji Medical College,Huazhong University of Science and Technology,Wuhan 430030 Hubei,China [2]Wuhan Institute of Medical Sciences,Wuhan 430014,China

出　　处：《Frontiers in Biology》2006年第1期56-60,共5页生物学前沿（英文版）

基　　金：supported by National Nature Science Foundation of China (No.30070722).

摘　　要：Tumor necrosis factor(TNF)-α-converting enzyme(TACE)is the major protease responsible for processing pro-TNF-αfrom membrane-anchored precursors to secreted TNF-α.In the present study,a 15-peptide library was used to identify potential TACE antagonists.To obtain the recombinant TACE ectodomain and to use it as a selective molecule for the screening of peptide inhibitors of TACE,cDNA coding for the catalytic domain(T800)and full-length ectodomain(T1300)of TACE were amplified by reverse transcription–polymerase chain reaction.The expression plasmid were constructed by inserting T800/T1300 into plasmid pET-28a/c respectively and were transformed into Escherichia coli BL21(DE3).Sodium dodecyl sulfate–polyacrylamide gel electrophoresis(SDSPAGE)andWestern blot analysis revealed that T800/T1300 were highly expressed in the form of an inclusion body induced by isopropylthiogalactoside.After Ni2+–NTA resin affinity chromatography,the purity of the recombinant T800/T1300 protein was more than 90%.T800 and T1300 proteins were used in the screening of T800/T1300-binding peptides from a phage display random 15-peptide library.After four rounds of biopanning,the positive phage clones were analyzed by enzyme-linked immunosorbent assay,competitive inhibition assay(ELESA),and DNA sequencing.A common amino acid sequence(TRWLVYFS RPYLVAT)was confirmed and synthesized.A synthetic peptide was shown to bind to TACE and to inhibit TNF-αrelease from lipopolysaccharide(LPS)-stimulated human peripheral blood mononuclear cells(PBMC)by up to 60.3%.Fluorescence-activated cell sorter(FACS)analysis revealed that the peptide mediated the accumulation of TNF-αon an LPS-stimulated PBMC surface.These results demonstrate that the TACE-binding peptide is an effective antagonist of TACE and that the deduced motif might be applied to the molecular design of anti-inflammatory drugs.

关 键 词：TACE ECTODOMAIN phage display random peptide library peptide inhibitor 

分 类 号：R73[医药卫生—肿瘤]