Biochim Biophys Acta Proteins Proteom 2019 Feb 28;1867(2):152-162. Epub 2018 Nov 28.
Department of Biochemistry, Science Institute, University of Iceland, Reykjavík, Iceland. Electronic address:
Cloning into a pET 11a vector, followed by high-level expression of the cold adapted subtilase, VPR, utilizing the rhamnose titratable T7 system of Lemo21, resulted in a dramatic increase of soluble protein compared to the older system used. Expression optimization clearly shows the importance of calcium in the medium after induction, both for stability of the proteinase and cell health. Characterization of the purified enzyme obtained in a redesigned purification protocol which removed apparent RNA contaminants, resulted in a significantly higher value for k than previously reported. The new recombinant protein exhibited slightly lower stability against thermal denaturation and thermal inactivation. Our results also indicate that two of the calcium binding sites have apparent binding constants in the mM range. Binding of calcium to the weaker of those two sites only affects resistance of the enzyme against irreversible thermal inactivation. Differential scanning calorimetry revealed a non-two-state denaturation process, with indication of presence of intermediates caused by unfolding of calcium binding motifs.