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., Eur. J. Biochem. 211 (1993) 305-310]. The P2-encoding synthetic gene was expressed in E. coli and in Saccharomyces cerevisiae. The recombinant (re-) protein was produced to approx. 1.5% of the total protein content in S. cerevisiae using the galactose-inducible GAL1 promoter and to 3% (tac/lac tandem promoters) or 6.5% (T7 promoter) in E. coli as judged by immunological and biochemical criteria. E. coli-produced 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 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 E. coli- 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 lysine methylation does not play a role in P2 thermostabilization.
Fusi
P
P
Dipartimento di Fisiologia e Biochimica Generali, Università degli Studi di Milano, Italy.
Grisa
M
M
Mombelli
E
E
Consonni
R
R
Tortora
P
P
Vanoni
M
M
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification; D001483:Base Sequence; D003001:Cloning, Molecular; D004926:Escherichia coli; D005813:Genes, Synthetic; D008745:Methylation; D008969:Molecular Sequence Data; D011499:Protein Processing, Post-Translational; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D012260:Ribonucleases / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification; D012441:Saccharomyces cerevisiae; D016415:Sequence Alignment; D013045:Species Specificity; D016944:Sulfolobus / Q000201:enzymology / Q000235:genetics*
D001426:Bacterial Proteins; D011993:Recombinant Fusion Proteins; D012260:Ribonucleases; C092231:ribonuclease P2