AMPLIFIED EXPRESSION, PURIFICATION AND FUNCTIONAL RECONSTITUTION OF THE DIPEPTIDE AND TRIPEPTIDE TRANSPORT PROTEIN OF LACTOCOCCUS-LACTIS

Transport of hydrophilic dipeptides and tripeptides into Lactococcus l actis is mediated by a protonmotive-force-driven peptide-transport pro tein (DtpT) that shares similarity to eukaryotic peptide transporters, e.g. from yeasts, plants, and the kidney and small intestine of rabbi t, man and rat. The expression level of DtpT protein in L. lactis was increased (20-40-fold) to approximately 10% of total integral membrane protein by means of a low-copy-number vector and selecting the approp riate growth conditions. Membrane vesicles bearing the DtpT-His(6) pro tein (containing a C-tenninal factor-Xa cleavage site and a six-histid ine-tag) showed a Pro-Ala uptake activity that was half that of membra nes containing the wild-type protein. The activity in the DtpT-His(6) membrane vesicles increased at least 50 % upon removal of the His(6) t ag from the protein. More than 95 % DtpT was solubilized from 'L. lact is membranes in the presence of 1 % (mass/vol.) n-dodecyl-beta-D-malto side: and approximately 2 mg DtpT-His, was purified by Ni2+-chelate af finity chromatography from 200 mg membrane protein. Purified DtpT-His, was reconstituted unidirectionally into detergent-saturated formed li posomes, which were prepared from Escherichia coli phospholipid and ph osphatidylcholine; the detergent was removed by adsorption to polystyr ene beads. The highest uptake activities were obtained when DtpT was i ncorporated into liposomes that were treated with a low amount of n-do decyl-beta-D-maltoside (onset of liposome solubilization). The uptake activity could be improved by addition of NaCl (200 mM) and lipids (2 mg/ml) during the solubilization, purification and reconstitution step s.