Screening of TACE peptide inhibitors from phage display peptide library.
To obtain the recombinant tumor necrosis factor-alpha converting enzyme (TACE) ectodomain and use it as a selective molecule for the screening of TACE peptide inhibitors, the cDNA coding catalytic domain (T800) and full-length ectodomain (T1300) of TACE were amplified by RT-PCR, and the expression plasmids were constructed by inserting T800 and T1300 into plasmid pET-28a and pET-28c respectively. The recombinant T800 and T1300 were induced by IPTG, and SDS-PAGE and Western blotting analysis results revealed that T800 and T1300 were highly expressed in the form of inclusion body. After Ni(2+)-NTA resin affinity chromatography, the recombinant proteins 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-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 the cell surface. These results demonstrate that the TACE-binding peptide is an effective antagonist of TACE.
Huang
Wei
W
Department of Biochemistry & Molecular Biology, School of Basic Medical Sciences, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, China.
Li
Lingbo
L
Han
Ling
L
Yang
Yuzhen
Y
D051722:ADAM Proteins / Q000037:antagonists & inhibitors* / Q000096:biosynthesis / Q000235:genetics; D000072198:ADAM17 Protein; D020134:Catalytic Domain; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005822:Genetic Vectors / Q000235:genetics; D006801:Humans; D007963:Leukocytes, Mononuclear / Q000166:cytology; D019151:Peptide Library; D010455:Peptides / Q000737:chemistry / Q000494:pharmacology; D011387:Prokaryotic Cells / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D014409:Tumor Necrosis Factor-alpha / Q000037:antagonists & inhibitors*
D019151:Peptide Library; D010455:Peptides; D011994:Recombinant Proteins; D014409:Tumor Necrosis Factor-alpha; D051722:ADAM Proteins; D000072198:ADAM17 Protein; C000606462:ADAM17 protein, human