Synthesis of peptide bonds by proteinases.

However, two published papers reported the successful expression of active NK and subtilisin DFE in E. coli 62, 63. Both papers took advantage of the principle that the extracellular protease subtilisin from the genus Bacillus is synthesized as Pre-pro enzymes, and its pro peptide may function as an intra-molecular chaperon to facilitate correct folding of protease domine 64, 65. Zhang et al 63 showed that substilisin DFE was highly expressed in E. coli BL21(DE3) as fusion protein of Trx-prosubtilisin DFE via the expression vector pE32a and that strong fibrinolytic activity was detected in both soluble fraction and inclusion bodies fraction after in vitro renaturation. Moreover, the fusion proteins are easily purified and refolded in a column to activate enzyme. Most importantly, Trx-peptides can be automatically cleaved during in vitro refolding to form the mature subtilisin DFE.

Solid-Phase synthesis of protected peptide

Enzymes in organic synthesis: use of

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.

Methods of synthesis and use are also comprised in the ..

The increased identification of phosphorylation pathways in many biochemical processes has necessitated a demand for the synthesis of phosphorylated peptides for use as substrates. Mimotopes has been at the forefront of developing reliable methods for the synthesis of phophopeptides for use in substrate mapping studies.

Why use Special Amino Acids in peptide synthesis

Reactivity of D-amino acid amides as acyl acceptors and partially unblocked amino acid acyl donors further expands the generality of the use of organic solvent-soluble enzymes as peptide synthesis catalysts.">

Automated solid-phase peptide synthesis to ..

Fluorescent donor and acceptor groups are quite large so spacer residues are usually incorporated into test sequences to ensure access to potential recognition sites. Hydrophilic spacers, such as b-alanine, are preferred as these make the peptides easier to solubilise. If a hydrophobic spacer has to be used, peptide solubility can be improved by the addition of one or more lysine residues at the C terminus.

Synthesis procedures are monitored closely to minimise the incidence of peptides labelled with only one or other of the fluorophores.

FRET peptides can be designed with or without prior knowledge of target sequences. Where information about targets is limited, a replacement set of amino acids can be defined for each residue position and all possible combinations prepared. If the replacement set is large or two or more residue positions are variable, mixtures can be used to reduce the number of peptides in the screening set.

Replacement sets can include L-amino acids, D-amino acids and any unusual amino acids, which are available commercially in an Fmoc-protected form.