Methods in Molecular Biology • 16 Enzymes of Molecular Biology

CHAPTER 15

Proteolytic Enzymes

for Peptide Production

Patricia J. Sweeney and John M. Walker

1. Introduction
   There are three main reasons why a protein chemist might wish to
   cleave a protein of interest into peptide fragments. The first reason is
   to generate, by extensive proteolysis, a large number of relatively
   small (5-20 residues) peptides either for peptide mapping (see vol. 1,
   Chapter 5) or for purification and subsequent manual sequence deter-
   mination by the dansyl-Edman method (see vol. 1, Chapter 24). The
   second reason is to generate relatively large peptides (50-150 resi-
   dues) by limited proteolysis for automated sequence analysis, such as
   with the gas-phase sequencer. The third reason is to prepare, again by
   limited proteolysis, specific fragments for studies relating structure to
   function. In each case, the specificity of the enzyme used to generate
   the peptides is a prime consideration, since the aim is to provide high
   yields of discrete fragments. It can be appreciated that significantly
   <100% cleavage at some or all of the cleavage sites on the protein
   being digested will generate a far more complex mixture of a larger
   number of polypeptides, each in relatively low yield. It is for this
   reason that enzymes of high specificity, such as trypsin, which cleaves
   at the C-terminal side of arginine and lysine residues, are mainly used
   for peptide production. However, other proteases with considerably
   less specificity have also found use in peptide production, particularly
   when limited proteolysis is being used, or where native protein is used
   as the substrate when only a limited number of susceptible peptide

From: Methods in Molecular Biology, Vet 16: Enzymes of Molecular Biology

Edited by: M. M. Burrell Copyright ©1993 Humana Press Inc., Totowa, NJ

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