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) fro m the enzyme transcarboxylase (EC 2.1.3.1) of Propioni shermanii was overex pressed in Escherichia coli. Complete biotinylation of the protein was achi eved by addition of exogenous biotin and coexpression of the biotin holoenz yme synthetase (EC 6.3.4.15.) from E. coli. The transcription of both genes was put under control of different operators/promoters, thus achieving ind ependent control of expression levels and optimized yields of the holo-tcc. Bacteria were grown in a biotin-supplemented minimal medium (M9) that cont ained [C-13]glucose as the carbon source and [N-15]NH4Cl as the sole nitrog en source. The target protein could be purified to homogeneity by ion-excha nge chromatography and concentrated to NMR-suitable concentrations (2 mM) w ithout aggregation. (C) 1999 Academic Press.