Purification of daptomycin binding proteins (DBPS) from the membrane of Enterococcus hirae

Daptomycin binding proteins (DBPs) are membrane proteins which act as dapto mycin targets. Daptomycin is a cyclic lipopeptide antibiotic which is activ e against Gram-positive bacteria and was shown to be the first inhibitor of lipoteichoic acid (LTA) synthesis. It was found that the antibiotic did no t penetrate the bacterial cytoplasm but bound membranes with a non-covalent bond and in particular some proteins which were called DBPs. DBPs were ind icated as enzymes involved in LTA synthesis whose binding and inhibition by daptomycin is responsible for the observed effect on bacterial LTA synthes is. The purification of DBPs will make it possible not only to shed light o n the biosynthesis of the cell wall polymer but will also provide innovativ e targets for selection of new antibacterial compounds. In this study, the purification of DBPs is described. Affinity chromatography was used with da ptomycin as the ligand. Final elution of DBPs from daptomycin-coupled resin was performed using either 0.1% SDS or 3 M NaCl. Polyacrylamide gel electr ophoresis of the eluted protein fractions consistently showed four protein bands (ranging from 55 to 66 kDa) in denaturating conditions and two protei n bands (60 and 66 kDa) in non-denaturating conditions. Isoelectrofocusing analysis of the same sample consistently revealed two bands with pIs around 5. That these purified proteins were really the desired DBPs is demonstrat ed by the retention of daptomycin-binding capability they displayed.