Unidirectional reconstitution and characterization of purified Na+/proline transporter of Escherichia coli
Autor(en): | Jung, H Tebbe, S Schmid, R Jung, K |
Stichwörter: | Biochemistry & Molecular Biology; EXPRESSION; GENES; IDENTIFICATION; LIPOSOMES; PERMEASE; PROLINE CARRIER; PROTEINS; SYMPORT CARRIER; SYSTEM; VECTORS | Erscheinungsdatum: | 1998 | Herausgeber: | AMER CHEMICAL SOC | Enthalten in: | BIOCHEMISTRY | Band: | 37 | Ausgabe: | 31 | Startseite: | 11083 | Seitenende: | 11088 | Zusammenfassung: | 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 [V-max = 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 Nai or Lif 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. |
ISSN: | 00062960 | DOI: | 10.1021/bi980684b |
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geprüft am 06.06.2024