PubMed · 4812342
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D D Bayse. Proficiency testing and consensus evaluation.. https://pubmed.ncbi.nlm.nih.gov/4812342/
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The gene for an archaebacterial hyperthermophilic enzyme, aspartate aminotransferase from Sulfolobus solfataricus (AspATSs), was expressed in Escherichia coli and the enzyme purified to homogeneity. A suitable expression vector and host strain were selected and culture conditions were optimized so that 6-7 mg of pure enzyme per litre of culture were obtained repeatedly. The recombinant enzyme and the authentic AspATSs are indistinguishable: in fact, they have the same molecular weight, estimated by means of SDS-PAGE and gel filtration, the same Km values for 2-oxo-glutarate and cysteine sulphinate and the same UV-visible spectra. Moreover, recombinant AspATSs is thermophilic and thermostable just as the enzyme extracted from Sulfolobus solfataricus. The protocol described may be used to produce thermostable arachaebacterial enzymes in mesophilic hosts.
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Equilibrium dissociation and unfolding of dimeric aspartate aminotransferase from Escherichia coli proceeds via two compact monomeric intermediates which have similar hydrodynamic volumes but different fluorescence properties. We probed binding of the coenzyme pyridoxal 5'-phosphate to these intermediates by coupling fluorescence detection to size-exclusion HPLC. This procedure gave additionally an internal conformational probe of the unfolding transitions of the enzyme. It was shown that the first intermediate, M, is able to bind the coenzyme, whereas the second intermediate, M*, is not. It is likely that M is the correctly folded monomer of the protein.