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Screening of Peptide Inhibitors of TACE from a Phage Display Random 15-Peptide Library by Recombinant TACE Ectodomain |
| Huang Wei1, Yang Yuzhen2, Wang Zhen2, Hang Ling2 |
| 1.Department of Biochemistry and Molecular Biology, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030 Hubei, China; Wuhan Institute of Medical Sciences, Wuhan 430014, China; 2.Department of Biochemistry and Molecular Biology, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030 Hubei, China |
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Abstract Tumor necrosis factor (TNF)-α-converting enzyme (TACE) is the major protease responsible for processing pro-TNF-α from 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-α release 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-α on 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.
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Issue Date: 05 March 2006
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