Screening of TACE peptide inhibitors from a phage display random 15-peptide library by recombinant TACE ecotodomain.
- Author:
Wei HUANG
1
;
Ling-Bo LI
;
Ling HAN
;
Hui ZHANG
;
Yu-Zhen YANG
Author Information
1. Department of Biochemistry & Molecular Biology, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, China.
- Publication Type:Journal Article
- MeSH:
ADAM Proteins;
antagonists & inhibitors;
biosynthesis;
genetics;
ADAM17 Protein;
Amyloid Precursor Protein Secretases;
Animals;
Humans;
Mice;
Peptide Library;
Peptides;
chemistry;
Recombinant Proteins;
biosynthesis;
genetics;
Tumor Necrosis Factor-alpha
- From:
Chinese Journal of Biotechnology
2005;21(1):30-35
- CountryChina
- Language:Chinese
-
Abstract:
Tumour necrosis factor-alpha converting enzyme (TACE) is the major protease responsible for processing proTNF from membrane-anchored precursor into secreted TNF-alpha. It was validated that TACE is involved in many diseases such as arthritis, multiple sclerosis and Alzheimers, therefore it represents a novel and significant target for therapeutic intervention in a variety of inflammatory and neuroimmunological diseases. To obtain the recombinant TACE ectodomain and use it as a selective molecule for the screening of TACE peptide inhibitors, the cDNA coded for catalytic domain (T800) and full-length ectodomain (T1300) of TACE were amplified by RT-PCR, the expression plasmid was constructed by inserting T800/T1300 into plasmid pET-28a/pET-28c and transformed into E. coli BL21 (DE3). SDS-PAGE and Western blotting analysis revealed that T800/T1300 was highly expressed in the form of inclusion body being induced by IPTG. After Ni2+ -NTA resin affinity chromatography, the purity of the recombinant T800/T1300 protein was more than 90%. T800 and T1300 protein were used in the screening of TACE-binding peptides from the phage display random 15-peptide library. After four 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 was shown to bind to TACE and inhibit the TNF-alpha 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-alpha on LPS-stimulated PBMC surface. These results demonstrate that the TACE-binding peptide is an effective antagonist of TACE and the deduced motif might be applied to molecular design of anti-inflammation drugs.
