Screening of TACE Peptide Inhibitors from Phage Display Peptide Library

• Published in: Journal of Huazhong University of Science and Technology. Medical sciences Vol. 25; no. 5; pp. 473 - 476
• Main Author: 黄巍 李凌波 韩玲 杨渝珍
• Format: Journal Article
• Language: English
• Published: China Department of Biochemistry & Molecular Biology, School of Basic Medical Sciences, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, China 2005; Wuhan Institute of Medical Sciences,Wuhan 430014,China%Department of Biochemistry & Molecular Biology, School of Basic Medical Sciences, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, China
• Subjects: ADAM Proteins - antagonists & inhibitors; ADAM Proteins - biosynthesis; ADAM Proteins - genetics; ADAM17 Protein; Catalytic Domain; Escherichia coli - genetics; Escherichia coli - metabolism; Genetic Vectors - genetics; Humans; Leukocytes, Mononuclear - cytology; Peptide Library; Peptides - chemistry; Peptides - pharmacology; Prokaryotic Cells - metabolism; Recombinant Proteins - biosynthesis; Recombinant Proteins - genetics; Tumor Necrosis Factor-alpha - antagonists & inhibitors; 图书馆; 外周血; 抗生素; 缩氨酸; 肿瘤坏死因子-α转化酶; tumor necrosis factor-α converting enzyme; phage display; ectodomain; prokaryote expression; peptide inhibitor
• ISSN: 1672-0733; 1993-1352
• DOI: 10.1007/BF02895991

Summary： To obtain the recombinant tumor necrosis factor-α converting enzyme （TACE） ectodomain and use it as a selective molecule for the screening of TACE peptide inhibitors, the cDNA coding catalytic domain （TS00） and full-length ectodomain （T1300） of TACE were amplified by RT-PCR, and the expres.sion plasmids were constructed by inserting T800 and T1300 into plasmid pET-28a and pET-28c respectively. The recombinant TS00 and T1300 were induced by IPTG, and SDS-PAGE and Western blotting analysis results revealed that TS00 and T1300 were highly expressed in the form of inclusion body. After Ni^2＋-NTA resin affinity chromatography, the recombinant proreins were used in the screening of TACE-binding peptides from phage display peptide library respectively. After 4 rounds of biopanning, the positive phage clones were analyzed by ELISA, competitive inhibition assay and DNA sequencing. A common amino acid sequence （TRWLVYFSRPYLVAT） was found and synthesized. The synthetic peptide could inhibit the TNF-α release from LPS-stimulated human peripheral blood mononuclear cells （PBMC） up to 60.3%. FACS analysis revealed that the peptide mediated the accumulation of TNF-α on the cell surface. These results demonstrate that the TACE binding peptide is an effective antagonist of TACE.