Amplified expression, purification and functional reconstitution of the dipeptide and tripeptide transport protein of Lactococcus lactis
1997
Hagting, A. | Knol, J. | Hasemeier, B. | Streutker, M.R. | Fang, G. | Poolman, B. | Konings, W.N.
Transport of hydrophilic dipeptides and tripeptides into Lactococcus lactis is mediated by a proton-motive-force-driven peptide-transport protein (DtpT) that shares similarity to eukaryotic peptide trans-porters, 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-His6 protein (containing a C-terminal 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-His6 membrane vesicles increased at least 50% upon removal of the His6 tag from the protein. More than 95% DtpT was solubilized from L. lactis membranes in the presence of 1% (mass/vol.) n-dolecyl-beta-n-maltoside, and approximately 2 mg DtpT-His6 was purified by Ni2+ -chelate affinity chromatography from 100 mg membrane protein. Purified DtpT-His6 was reconstituted unidirectionally into detergent-saturated formed liposomes, which were prepared from Escherichia coli phospholipid and egg 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 millimole) and lipids (2 mg/ml) during the solubilization, purification and reconstitution steps.
اظهر المزيد [+] اقل [-]الكلمات المفتاحية الخاصة بالمكنز الزراعي (أجروفوك)
المعلومات البيبليوغرافية
تم تزويد هذا السجل من قبل National Agricultural Library