Large scale purification, nucleotide binding properties, and ATPase activity of the MalK subunit of Salmonella typhimurium maltose transport complex.

The malK gene, encoding a membrane-associated component of the maltose transport complex of Salmonella typhimurium was cloned into an expression vector downstream of the promoters lambda pR and lambda pL and a strong translation initiation region. Escherichia coli strain JM109 harboring the resulting plasmid pCW14 synthesized a protein of apparent molecular mass of 43 kDa upon temperature shift, as demonstrated by sodium dodecyl sulfate-gel electrophoresis. The identity of the protein was determined by N-terminal amino acid sequencing. The overproduced protein was sequestered in inclusion bodies as revealed by electron microscopy. The protein was purified to homogeneity on a large scale by disrupting the cells with a passage through a Ribi press, solubilizing the inclusion bodies with urea, and subsequent chromatography on Red Agarose. Purified MalK, as the membrane-bound MalK protein could be covalently modified by [gamma-32P]8-azido-ATP. Furthermore, the purified protein bound [gamma-32P] ATP with a dissociation constant of 150 microM and exhibited ATPase activity, which was stimulated by dimethyl-sulfoxide and inhibited by ADP.