Amplified expression, purification and functional reconstitution of the dipeptide and tripeptide transport protein of Lactococcus lactis

Transport of hydrophilic dipeptides and tripeptides into Lactococcus lactis is mediated by a protonmotive-force-driven peptide-transport protein (DtpT) that shares similarity to eukaryotic peptide transporters, e.g. from yeasts, plants, and the kidney and small intestine of rabbit, 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 appropriate growth conditions. Membrane vesicles bearing the DtpT-His(6) protein (containing a C-tenninal factor-Xa cleavage site and a six-histidine-tag) showed a Pro-Ala uptake activity that was half that of membranes containing the wild-type protein. The activity in the DtpT-His(6) membrane vesicles increased at least 50 % upon removal of the His(6) tag from the protein. More than 95 % DtpT was solubilized from 'L. lactis membranes in the presence of 1 % (mass/vol.) n-dodecyl-beta-D-maltoside: and approximately 2 mg DtpT-His, was purified by Ni2+-chelate affinity chromatography from 200 mg membrane protein. Purified DtpT-His, was reconstituted unidirectionally into detergent-saturated formed liposomes, which were prepared from Escherichia coli phospholipid and phosphatidylcholine; the detergent was removed by adsorption to polystyrene beads. The highest uptake activities were obtained when DtpT was incorporated into liposomes that were treated with a low amount of n-dodecyl-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 steps.