Heterologous expression of 5 '-methylthioadenosine phosphorylase from the archaeon Sulfolobus solfataricus: Characterization of the recombinant protein and involvement of disulfide bonds in thermophilicity and thermostability

The gene for the extremely thermophilic and thermostable 5'-methylthioadeno sine phosphorylase from the archaeon Sulfolobus solfataricus was expressed at a high level in Escherichia coli thus providing a basis for detailed str uctural and functional studies of the enzyme. The recombinant enzyme was pu rified to homogeneity by means of a heat treatment (10 min at 100 degrees C ) and by a single affinity chromatography step. The appropriate expression vector and host strain were selected and the culture conditions were determ ined that would ensure a consistent yield of 6 mg of pure enzyme per liter of culture. The heterologously expressed enzyme is identical to the origina l S. solfataricus 5'-methylthioadenosine phosphorylase regarding molecular weight, substrate specificity, and the presence of intersubunit disulfide b onds. On the other hand, the recombinant 5'-methylthioadenosine phosphoryla se is less thermophilic and thermostable than the S. solfataricus enzyme, s ince an incorrect positioning of disulfide bonds within the molecule genera tes structures less stable to thermal unfolding. (C) 1999 Academic Press.