Solid-Phase synthesis of protected peptide
Use of enzymes in peptide synthesis - [PDF Document]
Identification of peptide substrates for proteases can be a major undertaking. To overcome issues such as feasibility and deconvolution, associated with large peptide libraries, Mimotopes has developed in collaboration with GSK a ‘small but smart’ generic fluorescence resonance energy transfer rapid endopeptidase profiling library () as a tool for rapidly identifying protease substrates. Within a tripeptide core, flanked by Gly residues, similar amino acids were paired giving rise to a relatively small library of 3375 peptides divided into 512 distinct pools each containing only 8 peptides. The REPLi has been validated with trypsin, pepsin, the matrix metalloprotease (MMP)-12 and MMP-13 and calpains-1 and -2. In the case of calpain-2, a single iteration step involving LC-MS, provided the definitive residue specificity from which a highly sensitive fluorogenic substrate was then designed. The thorough validation of this ‘small but smart’ peptide library with representatives from each of the four mechanistic protease classes indicates that the REPLi will be useful for the rapid identification of substrates for multiple proteases.
PepSets REPLi is presynthesized in two scales (5nmol and 50nmol) and ready to ship. View REPLi specifications in further detail.
Use of enzymes in peptide synthesis | SpringerLink
FRET peptides are typically labelled with a fluorescent donor and a non-fluorescent acceptor. When these molecules form part of the same short peptide, they are close enough for the acceptor to quench the signal from the donor and for there to be very little fluorescence emission. However, if the acceptor is removed - for example, by protease cleavage - the quenching effect is lost, leading to increased fluorescence from the donor at the appropriate excitation wavelength.
FRET peptides can therefore be used to study any biochemical reaction, which changes the physical distance between donor and acceptor molecules. FRET assays offer a safer alternative to the use of radiolabelled isotopes and methods are quick, sensitive and easily automated.