SUBTILISIN-CATALYZED RELIGATION OF PROTEOLYZED HEN EGG-WHITE LYSOZYME- INVESTIGATION OF THE ROLE OF DISULFIDES

Background: The use of proteases to form, instead of break, peptide bo nds has expanded the repertoire of techniques available for protein se misynthesis. Several groups have previously reported the use of protea ses in aqueous-organic solvents to form single amide bonds within prot eins, but low yields and lengthy reaction times make this an impractic al approach to protein synthesis. We recently found that proteolyzed t riose 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 f rom a mixture of its proteolyzed fragments in high yield and with rapi d reaction times. This enzymatic religation can also be achieved after reduction of the four disulfide bonds present in lysozyme, with the s ame efficiency as that observed for the disulfide-containing proteolys is mixture. Conclusions: For egg-white lysozyme, the subtilisin religa tion reaction can be used to re-synthesize a proteolyzed protein even after reduction of disulfide bonds. The utility of this reaction in mo re generalized protein semisynthesis reactions is currently being expl ored.