EXPRESSION OF A SYNTHETIC GENE ENCODING P2 RIBONUCLEASE FROM THE EXTREME THERMOACIDOPHILIC ARCHAEBACTERIUM SULFOLOBUS-SOLFATARICUS IN MESOPHYLIC HOSTS

This work reports the molecular cloning and expression of a synthetic gene encoding P2, a 7-kDa ribonuclease (RNase) previously isolated in our laboratory from the archaebacterium Sulfolobus solfataricus [Fusi et al., fur. J. Biochem. 211 (1993) 305-310]. The P2-encoding syntheti c gene was expressed in E. coli and in Saccharomyces cerevisiae. The r ecombinant (re-) protein was produced to approx. 1.5% of the total pro tein content in S. cerevisiae using the galactose-inducible GAL1 promo ter and to 3% (tac/lac tandem promoters) or 6.5% (17 promoter) in E. c oli as judged by immunological and biochemical criteria. E. coli-produ ced P2 was purified to electrophoretic homogeneity through a one-step procedure, i.e., DEAE-Sephacel chromatography at pH 9.3, S. cerevisiae -produced P2 additionally required filtration through a Centricon-10 m icroconcentrator to obtain the same purity. The re-P2 was found to be indistinguishable from the Su. solfataricus enzyme on the basis of hea t stability, pH optimum and RNA digestion pattern. Furthermore, monodi mensional nuclear magnetic resonance showed that the E. coli- and Su. solfataricus-produced enzymes were structurally identical, the only ex ceptions being that Lys(4) and Lys(6) were not methylated in the re-en zyme,thus showing that lysine methylation does not play a role in P2 t hermostabilization.