Expression of a synthetic gene encoding P2 ribonuclease from the extreme thermoacidophilic archaebacterium XXXX in mesophylic hosts.

@entity11870 @entity12204 @entity3649 @entity8851 @entity376 @entity375 @entity526
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 @entity12204 [Fusi et al., Eur. J. Biochem. 211 (1993) 305-310]. The P2-encoding synthetic gene was expressed in @entity526 and in @entity375 . The recombinant (re-) protein was produced to approx. 1.5% of the total protein content in @entity375 using the @entity3649 -inducible @entity11870 promoter and to 3% (tac/lac tandem promoters) or 6.5% (T7 promoter) in @entity526 as judged by immunological and biochemical criteria. @entity526 -produced P2 was purified to electrophoretic homogeneity through a one-step procedure, i.e., @entity8851 -Sephacel chromatography at pH 9.3. @entity375 -produced P2 additionally required filtration through a Centricon-10 microconcentrator to obtain the same purity. The re-P2 was found to be indistinguishable from the Su. solfataricus enzyme on the basis of heat stability, pH optimum and RNA digestion pattern. Furthermore, monodimensional nuclear magnetic resonance showed that the @entity526 - and Su. solfataricus-produced enzymes were structurally identical, the only exceptions being that Lys4 and Lys6 were not methylated in the re-enzyme, thus showing that @entity376 methylation does not play a role in P2 thermostabilization.
