Unidirectional reconstitution and characterization of purified Na+/proline transporter of Escherichia coli.
A simple approach for large-scale purification and unidirectional reconstitution of the Na+/proline transporter of Escherichia coli (PutP) is described. The procedure is based on the insertion of a highly polar peptide composed of 17 amino acids including a 6His tag at the C-terminus of the transporter. Purification of the hybrid protein is achieved by Ni+-NTA affinity (purity >95%) and ion exchange chromatography (purity >99%). The purified transporter is reconstituted into preformed, detergent-destabilized liposomes. Detergent is removed slowly by adsorption to polystyrene beads. The highest activities [Vmax = 1.1 x 10(3) nmol min-1 (mg of protein)-1] are measured when Triton X100 is used for liposomes destabilization at a concentration corresponding to the onset of lipid solubilization. Site-directed labeling of PutP and site-specific proteolytic cleavage indicate that the transporter is inserted into proteoliposomes in an inside-out orientation. Reconstituted PutP is able to accumulate proline against a concentration gradient in the presence of an inwardly directed electrochemical Na+ or Li+ gradient, while a pH gradient does not affect transport. The apparent proline affinity of PutP in proteoliposomes is similar to the value determined with intact cells. Interestingly however, the apparent Na+ affinity of reconstituted PutP is reduced by a factor of about 25 compared to cells, suggesting a lower cation affinity on the cytosolic side of PutP relative to the outside.
Jung
H
H
Universität Osnabrück, Fachbereich Biologie/Chemie, Arbeitsgruppe Mikrobiologie, Osnabrück, Germany. Jung_H@mail.biologie.uni-osnabrueck.de
Tebbe
S
S
Schmid
R
R
Jung
K
K
D026921:Amino Acid Transport Systems, Neutral; D001692:Biological Transport; D002846:Chromatography, Affinity; D002852:Chromatography, Ion Exchange; D003902:Detergents; D004926:Escherichia coli / Q000201:enzymology*; D026901:Membrane Transport Proteins / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D009391:Nephelometry and Turbidimetry; D017830:Octoxynol; D011392:Proline / Q000378:metabolism*; D011510:Proteolipids / Q000378:metabolism; D012964:Sodium / Q000378:metabolism*; D012995:Solubility
D026921:Amino Acid Transport Systems, Neutral; D003902:Detergents; D026901:Membrane Transport Proteins; D011510:Proteolipids; C020485:proteoliposomes; D017830:Octoxynol; C033082:proline transporter; D011392:Proline; D012964:Sodium