Use of intein-directed peptide biosynthesis to improve serum stability and bioactivity of a gelatinase inhibitory peptide.
Screening of phage display libraries allows rapid identification of peptides binding to a target. However, functional analysis of the phage sequences and their reproduction as soluble and stable peptides are often the most time-consuming part in the screening. We have used here intein-based peptide biosynthesis to produce a phage-display derived gelatinase inhibitory peptide CTTHWGFTLC and to identify the critical residues for gelatinase inhibitory activity by performing alanine-scanning mutagenesis. By biosynthetic incorporation of 5-fluorotryptophan, we obtained an inhibitor of MMP-2 and MMP-9 gelatinases that showed a 6-fold enhancement in serum stability in comparison to the wild-type peptide. The new peptide also had an improved ability to inhibit tumor cell migration. These studies indicate the utility of intein methodology for synthesis and design of peptides obtained by phage display.
Björklund
Mikael
M
Department of Biosciences, Division of Biochemistry, Viikinkaari 5, Viikki Biocenter, University of Helsinki, FIN-00014, Finland.
Valtanen
Heli
H
Savilahti
Harri
H
Koivunen
Erkki
E
D000409:Alanine / Q000235:genetics / Q000378:metabolism; D000595:Amino Acid Sequence; D002465:Cell Movement / Q000187:drug effects; D004355:Drug Stability; D004791:Enzyme Inhibitors / Q000097:blood* / Q000378:metabolism / Q000494:pharmacology*; D018093:Gelatinases / Q000037:antagonists & inhibitors*; D006801:Humans; D016254:Mutagenesis, Insertional / Q000379:methods; D019151:Peptide Library; D010455:Peptides / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology*; D019154:Protein Splicing; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology; D014364:Tryptophan / Q000031:analogs & derivatives / Q000378:metabolism; D014407:Tumor Cells, Cultured
D004791:Enzyme Inhibitors; D019151:Peptide Library; D010455:Peptides; D011993:Recombinant Fusion Proteins; D014364:Tryptophan; D018093:Gelatinases; D000409:Alanine