Expression and biotinylation of a mutant of the transcarboxylase carrier protein from Propioni shermanii.

A deletion mutant (residues 10 to 48 cut) of the biotinyl subunit (tcc) from the enzyme transcarboxylase (EC 2.1.3.1) of Propioni shermanii was overexpressed in Escherichia coli. Complete biotinylation of the protein was achieved by addition of exogenous biotin and coexpression of the biotin holoenzyme synthetase (EC 6.3. 4.15.) from E. coli. The transcription of both genes was put under control of different operators/promoters, thus achieving independent control of expression levels and optimized yields of the holo-tcc. Bacteria were grown in a biotin-supplemented minimal medium (M9) that contained [(13)C]glucose as the carbon source and [(15)N]NH(4)Cl as the sole nitrogen source. The target protein could be purified to homogeneity by ion-exchange chromatography and concentrated to NMR-suitable concentrations (2 mM) without aggregation.