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. 2013 Nov 8;3(4):923-42.
doi: 10.3390/biom3040923.

Research applications of proteolytic enzymes in molecular biology

János András Mótyán  1 , Ferenc Tóth  2 , József Tőzsér  3
Affiliations

Research applications of proteolytic enzymes in molecular biology

János András Mótyán et al. Biomolecules. .

Abstract

Proteolytic enzymes (also termed peptidases, proteases and proteinases) are capable of hydrolyzing peptide bonds in proteins. They can be found in all living organisms, from viruses to animals and humans. Proteolytic enzymes have great medical and pharmaceutical importance due to their key role in biological processes and in the life-cycle of many pathogens. Proteases are extensively applied enzymes in several sectors of industry and biotechnology, furthermore, numerous research applications require their use, including production of Klenow fragments, peptide synthesis, digestion of unwanted proteins during nucleic acid purification, cell culturing and tissue dissociation, preparation of recombinant antibody fragments for research, diagnostics and therapy, exploration of the structure-function relationships by structural studies, removal of affinity tags from fusion proteins in recombinant protein techniques, peptide sequencing and proteolytic digestion of proteins in proteomics. The aim of this paper is to review the molecular biological aspects of proteolytic enzymes and summarize their applications in the life sciences.

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Figures

Figure 1
Figure 1
Action of aminopeptidases and carboxypeptidases removing the terminal amino acid residues as well as endopeptidases on a polypeptide substrate (having n residues). Red arrows show the peptide bonds to be cleaved.
Figure 2
Figure 2
Kinetically-controlled synthesis of Z-d-Leu-l-Leu-NH2 dipeptide. After the formation of enzyme-substrate complex (K1) a covalent enzyme-substrate intermediate is formed (K2). The intermediate is subjected to the attack from H2O or other nucleophiles (Nu). KH is the equilibrium constant of hydrolysis, KT is the equilibrium constant of the transferase reaction.
Figure 3
Figure 3
Structure of IgG antibody molecules (A) and fragments released after proteolytic digestion using papain (B), pepsin (C) or ficin (D).
Figure 4
Figure 4
Steps of proteomic analysis using mass-spectrometry after separation and in-gel digestion of proteins of interest.
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