title: Subtilisin-catalyzed religation of proteolyzed hen egg-white lysozyme: investigation of the role of disulfides.
abstract: BACKGROUND
  The use of proteases to form, instead of break, peptide bonds has expanded the repertoire of techniques available for protein semisynthesis. Several groups have previously reported the use of proteases in aqueous-organic solvents to form single amide bonds within proteins, but low yields and lengthy reaction times make this an impractical approach to protein synthesis. We recently found that proteolyzed triose phosphate isomerase can be re-ligated rapidly and efficiently by subtilisin, in mixed aqueous-organic solvent systems.
  
  
  RESULTS
  We now report the use of subtilisin to resynthesize hen egg-white lysozyme from a mixture of its proteolyzed fragments in high yield and with rapid reaction times. This enzymatic religation can also be achieved after reduction of the four disulfide bonds present in lysozyme, with the same efficiency as that observed for the disulfide-containing proteolysis mixture.
  
  
  CONCLUSIONS
  For egg-white lysozyme, the subtilisin religation reaction can be used to re-synthesize a proteolyzed protein even after reduction of disulfide bonds. The utility of this reaction in more generalized protein semisynthesis reactions is currently being explored.
authors.affiliation: Department of Chemistry, Purdue University, West Lafayette, IN 47907, USA.
authors.forename: K
authors.initials: K
authors.lastname: Vogel
authors.forename: J
authors.initials: J
authors.lastname: Cook
authors.forename: J
authors.initials: J
authors.lastname: Chmielewski
mesh_terms: D000818:Animals; D002384:Catalysis; D002645:Chickens; D004220:Disulfides / Q000378:metabolism*; D004529:Egg White; D006868:Hydrolysis; D009113:Muramidase / Q000737:chemistry / Q000378:metabolism*; D011487:Protein Conformation; D017510:Protein Folding; D013381:Subtilisins / Q000378:metabolism*
chemical_list: D004220:Disulfides; D009113:Muramidase; D013381:Subtilisins