Heterologous expression of 5'-methylthioadenosine phosphorylase from the archaeon Sulfolobus solfataricus: characterization of the recombinant protein and involvement of disulfide bonds in thermophilicity and thermostability.
The gene for the extremely thermophilic and thermostable 5'-methylthioadenosine phosphorylase from the archaeon Sulfolobus solfataricus was expressed at a high level in Escherichia coli thus providing a basis for detailed structural and functional studies of the enzyme. The recombinant enzyme was purified to homogeneity by means of a heat treatment (10 min at 100 degrees C) and by a single affinity chromatography step. The appropriate expression vector and host strain were selected and the culture conditions were determined that would ensure a consistent yield of 6 mg of pure enzyme per liter of culture. The heterologously expressed enzyme is identical to the original S. solfataricus 5'-methylthioadenosine phosphorylase regarding molecular weight, substrate specificity, and the presence of intersubunit disulfide bonds. On the other hand, the recombinant 5'-methylthioadenosine phosphorylase is less thermophilic and thermostable than the S. solfataricus enzyme, since an incorrect positioning of disulfide bonds within the molecule generates structures less stable to thermal unfolding.
Cacciapuoti
G
G
Istituto di Biochimica delle Macromolecole, Facoltà di Medicina e Chirurgia, Seconda Università degli Studi di Napoli, Via Costantinopoli 16, Naples, 80138, Italy.
Fusco
S
S
Caiazzo
N
N
Zappia
V
V
Porcelli
M
M
D001483:Base Sequence; D002850:Chromatography, Gel; D004274:DNA, Recombinant / Q000235:genetics; D004220:Disulfides / Q000737:chemistry; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D019766:Genes, Archaeal; D008970:Molecular Weight; D010957:Plasmids / Q000235:genetics; D011683:Purine-Nucleoside Phosphorylase / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity; D016944:Sulfolobus / Q000201:enzymology* / Q000235:genetics*; D013696:Temperature
D004274:DNA, Recombinant; D004220:Disulfides; D011994:Recombinant Proteins; D011683:Purine-Nucleoside Phosphorylase; C021784:5'-methylthioadenosine phosphorylase