Screening of TACE peptide inhibitors from a phage display random 15-peptide library by recombinant TACE ecotodomain / 生物工程学报
Chinese Journal of Biotechnology
;
(12): 30-35, 2005.
Article
in Chinese
| WPRIM
| ID: wpr-256116
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.
Full text:
Available
Index:
WPRIM (Western Pacific)
Main subject:
Peptides
/
Recombinant Proteins
/
Chemistry
/
Tumor Necrosis Factor-alpha
/
Peptide Library
/
ADAM Proteins
/
Amyloid Precursor Protein Secretases
/
ADAM17 Protein
/
Genetics
Type of study:
Controlled clinical trial
/
Diagnostic study
/
Screening study
Limits:
Animals
/
Humans
Language:
Chinese
Journal:
Chinese Journal of Biotechnology
Year:
2005
Type:
Article
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