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 proton-motive-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-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-dodecyl-beta-D-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 mM) and lipids (2 mg/ml) during the solubilization, purification and reconstitution steps.
Hagting
A
A
Groningen Biomolecular Sciences and Biotechnology Institute, Department of Microbiology, University of Groningen, Haren, The Netherlands.
Knol
J
J
Hasemeier
B
B
Streutker
M R
MR
Fang
G
G
Poolman
B
B
Konings
W N
WN
D000818:Animals; D001426:Bacterial Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism*; D002352:Carrier Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism*; D003001:Cloning, Molecular; D004151:Dipeptides / Q000378:metabolism; D006801:Humans; D007421:Intestine, Small / Q000378:metabolism; D007668:Kidney / Q000378:metabolism; D007700:Kinetics; D013294:Lactococcus lactis / Q000378:metabolism*; D026901:Membrane Transport Proteins; D016297:Mutagenesis, Site-Directed; D009842:Oligopeptides / Q000378:metabolism*; D010944:Plants / Q000378:metabolism; D011817:Rabbits; D051381:Rats; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000378:metabolism; D016324:Sequence Tagged Sites
D001426:Bacterial Proteins; D002352:Carrier Proteins; D004151:Dipeptides; D026901:Membrane Transport Proteins; D009842:Oligopeptides; D011994:Recombinant Proteins; C086845:dtpT protein, Lactococcus lactis