Semisynthesis of carboxy-terminal fragments of thermolysin.
Enzyme-catalyzed synthesis of two polypeptide fragments, one of which is obtained by chemical synthesis, in the presence of proteolytic enzymes and in aqueous organic solvents constitutes a convenient procedure for the synthesis of proteins and their analogs. This novel semisynthetic procedure was investigated for preparing COOH-terminal fragments of the metallo-protease thermolysin. Fragment 205-316, obtained by autolysis of the protein in the presence of EDTA, was first cleaved selectively with Staphylococcus aureus V8 protease at the level of the single Glu302 residue into fragments 205-302 and 303-316. Upon incubation for 2-5 days of fragment 205-302 with a 5-fold excess of peptide 303-316, prepared by solid phase synthesis, with V8-protease in 0.1 M ammonium acetate, pH 6.0, containing 50% glycerol as organic cosolvent, enzyme-catalyzed reformation of the peptide bond was achieved in yields up to approximately 90% (based on fragment 205-302). The same procedure was used to prepare also the thermolysin fragments 205-315 and 205-311 by enzymatic coupling of fragment 205-302 to peptide 303-315 or 303-311, these last prepared by proteolytic digestion of the synthetic peptide 303-316. This procedure of semisynthesis opens up an approach for the site-directed modification of the tetrahelical COOH-terminal fragment 205-316 of thermolysin at the level of its helical segment encompassing residues 301-312 in the native, intact protein. Such analogs will be useful for examining structure-folding-stability relationships in this folded fragment possessing domain-like characteristics.
De Filippis
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Department of Organic Chemistry, Biopolymer Research Centre of CNR, University of Padua, Italy.
Fontana
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D000595:Amino Acid Sequence; D043424:Carboxypeptidase B; D002268:Carboxypeptidases / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid; D004492:Edetic Acid; D006358:Hot Temperature; D007700:Kinetics; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000138:chemical synthesis* / Q000378:metabolism; D011489:Protein Denaturation; D012697:Serine Endopeptidases / Q000378:metabolism; D013820:Thermolysin / Q000138:chemical synthesis* / Q000378:metabolism
D010446:Peptide Fragments; D004492:Edetic Acid; D002268:Carboxypeptidases; D043424:Carboxypeptidase B; D012697:Serine Endopeptidases; C028344:glutamyl endopeptidase; D013820:Thermolysin