Purification and functional characterization of the C-terminal half of the lactose permease of Escherichia coli

Biochemistry
J WuH R Kaback

Abstract

The lactose permease has been expressed in contiguous, non-overlapping polypeptide fragments containing the N-terminal (N6) and C-terminal (C6) transmembrane domains of the protein [Bibi, E., & Kaback, H. R. (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 4325; Zen, K., et al. (1994) Biochemistry 33, 8198]. When expressed individually, N6 and C6 are unstable and do not catalyze active transport. However, when expressed simultaneously, the polypeptides stabilize each other and form a complex that catalyzes active lactose transport. Moreover, a deletion construct containing the first transmembrane domain and the six C-terminal transmembrane domains mediates downhill lactose translocation [Bibi et al. (1991) proc. Natl. Acad. Sci. U.S.A. 88, 7271]. Here we report that C6 can be expressed independently in a relatively stable form that binds monoclonal antibodies 4B1 and 4B11, which interact with conformationally dependent epitopes on the periplasmic and cytoplasmic surfaces of the membrane, respectively. In addition, C6 retains the ability to catalyze lactose translocation down a concentration gradient in a specific manner. Finally, as observed with full-length Val331Cys permease, beta-D-galactopyranosyl 10thio-beta-D-galactopyranoside qu...Continue Reading

References

Aug 8, 1994·FEBS Letters·W WrubelR Ehring

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Citations

Dec 10, 1996·Proceedings of the National Academy of Sciences of the United States of America·E M Landau, J P Rosenbusch
Jan 29, 2002·Journal of Molecular Biology·Adam B WeinglassH Ronald Kaback
Feb 1, 1997·Protein Science : a Publication of the Protein Society·S FrillingosH R Kaback
Aug 15, 1997·The Journal of Biological Chemistry·M Panayotova-HeiermannE M Wright

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