Abstract:
: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.
journal_name
Protein Expr Purifjournal_title
Protein expression and purificationauthors
Cacciapuoti G,Fusco S,Caiazzo N,Zappia V,Porcelli Mdoi
10.1006/prep.1999.1076keywords:
subject
Has Abstractpub_date
1999-06-01 00:00:00pages
125-35issue
1eissn
1046-5928issn
1096-0279pii
S1046-5928(99)91076-8journal_volume
16pub_type
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