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 w as utilized as the initiation codon. This construct was used to transform E , coli RB791 and E, coli JM105 strains, The recombinant protein, purified b y a fast and efficient two-step procedure (yield of 0.4 mg of enzyme per gr am of cells), does not appear homogeneous on SDS-PAGE because of the presen ce of a protein contaminant corresponding to a "truncated" S-adenosylhomocy steine hydrolase subunit lacking the first 24 amino acid residues. The reco mbinant enzyme shows the same molecular mass, optimum temperature, and kine tic features of S-adenosylhomocysteine hydrolase isolated from S. solfatari cus but it is less thermostable. To construct a vector which presents a cor rect distance between the ribosome-binding site and the start codon of S-ad enosylhomocysteine hydrolase gene, a NcoI site was created at the translati on initiation codon using site-directed mutagenesis. The expression of the homogeneous mutant S-adenosylhomocysteine hydrolase was achieved at high le vel (1.7 mg of mutant protein per gram of cells), The mutant S-adenosylhomo cysteine hydrolase and the native one were indistinguishable in all physico chemical and kinetic properties including thermostability, indicating that the interactions involving the NH2-terminal sequence of the protein play a role in the thermal stability of S, solfataricus S-adenosylhomocysteine hyd rolase. (C) 2000 Academic Press.