Expression, purification, and characterization of recombinant S-adenosylhomocysteine hydrolase from the thermophilic archaeon Sulfolobus solfataricus.
S-Adenosylhomocysteine hydrolase from Sulfolobus solfataricus was expressed in Escherichia coli by inserting the genomic fragment containing the gene encoding for S-adenosylhomocysteine hydrolase downstream the isopropyl-beta-d-thiogalactoside-inducible promoter of pTrc99A expression vector. An ATG positioned 25 bp upstream of the gene which is in frame with a stop codon was utilized as the initiation codon. This construct was used to transform E. coli RB791 and E. coli JM105 strains. The recombinant protein, purified by a fast and efficient two-step procedure (yield of 0.4 mg of enzyme per gram of cells), does not appear homogeneous on SDS-PAGE because of the presence of a protein contaminant corresponding to a "truncated" S-adenosylhomocysteine hydrolase subunit lacking the first 24 amino acid residues. The recombinant enzyme shows the same molecular mass, optimum temperature, and kinetic features of S-adenosylhomocysteine hydrolase isolated from S. solfataricus but it is less thermostable. To construct a vector which presents a correct distance between the ribosome-binding site and the start codon of S-adenosylhomocysteine hydrolase gene, a NcoI site was created at the translation initiation codon using site-directed mutagenesis. The expression of the homogeneous mutant S-adenosylhomocysteine hydrolase was achieved at high level (1.7 mg of mutant protein per gram of cells). The mutant S-adenosylhomocysteine hydrolase and the native one were indistinguishable in all physicochemical and kinetic properties including thermostability, indicating that the interactions involving the NH(2)-terminal sequence of the protein play a role in the thermal stability of S. solfataricus S-adenosylhomocysteine hydrolase.
Porcelli
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M
Istituto di Biochimica delle Macromolecole, Facoltà di Medicina e Chirurgia, Seconda Università degli Studi di Napoli, Via Costantinopoli 16, Naples, 80138, Italy.
Fusco
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S
Inizio
T
T
Zappia
V
V
Cacciapuoti
G
G
D043383:Adenosylhomocysteinase; D001483:Base Sequence; D017931:DNA Primers / Q000235:genetics; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D019766:Genes, Archaeal; D006867:Hydrolases / Q000235:genetics* / Q000302:isolation & purification* / Q000378:metabolism; D007700:Kinetics; D008970:Molecular Weight; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010957:Plasmids / Q000235:genetics; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D016944:Sulfolobus / Q000201:enzymology* / Q000235:genetics*; D013696:Temperature
D017931:DNA Primers; D011994:Recombinant Proteins; D006867:Hydrolases; D043383:Adenosylhomocysteinase