Increase in Urea-Resistance of Recombinant V8 Protease by Combining Mutations, and Its Application in the Releasing of a Peptide Hormone from a Fusion Protein

概要

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In order to obtain a highly urea-resistant Staphylococcus aureus V8 protease for producing peptide hormones by recombinant DNA technology, we constructed double and triple mutants of recombinant S. aureus V8 protease by combining previously isolated urea-resistant mutations D44E, N71S and K147R. The double mutants were more stable than the single mutants, and the triple mutant was even more stable than the double mutants, showing that the effect caused by the three mutations was additive. The half life of the triple mutant (V8Δ53-U158) in 5 M urea was 21 times longer than that of S. aureus V8 protease. This increased stability by combining each urea-resistant mutation was only effective to urea, and not to 0.1% sodium dodecyl sulfate nor to heat at 50℃. The triple mutant was applied to the digestion reaction of a fusion protein containing the human calcitonin precursor peptide (hCT[G]). It was demonstrated that the fusion protein was efficiently digested in the presence of over 5 M of urea, and that the triple mutant could release 2 times more hCT[G] than could S. aureus V8 protease.
公益社団法人日本生物工学会の論文
1995-11-25