Monitoring channel activities of proteoliposomes with SecA and Cx26 gap junction in single oocytes
Section snippets
Protein purification
Escherichia coli SecA from BL21(λDE3)/pT7-SecA was purified as described previously [28]. SecA homologous from other bacteria was purified similarly from BL21.19 as described previously [10], [28], [29]. Purified proOmpA, an outer membrane protein precursor, was prepared as described previously [30], and membrane complexes of SecYEG and SecDF•YajC were purified as described previously [26].
Mouse gap junction protein Cx26 was expressed in insect cell line Sf9 using the Bac-to-Bac baculovirus
Bacterial protein-conducting channels with SecA–liposomes in oocytes
As described previously [23], by injecting oocytes with proteoliposomes together with essential factors such as ATP–Mg2+ and precursor proOmpA, we were able to detect increased outward currents with E. coli SecA–liposomes (Fig. 1 and Fig. 2A; the recording activities are presented in Fig. 1, upper left panel). Liposomes injected with purified SecA homologs from other bacteria, including Bacillus subtilis (Bs SecA), Bacillus anthracis (Ba SecA1), Staphylococcus aureus (Sa SecA1), Streptococcus
Discussion
In this article, we have combined several previously described methodologies for a simple and efficient system to correlate results with in vitro assembled protein complexes in liposomes to their in-cell functionality. This method employs direct injection of preassembled/folded proteins into proteoliposomes (Fig. 1) instead of cDNA or mRNA into Xenopus oocytes. Le Caherec and coworkers [35] described the injection of the reconstituted proteoliposomes into oocytes in order to monitor aquaporin
Acknowledgments
We thank D. Oliver, A. Driessen, F. Duong, and E. Lin for plasmids; R. Veenstra and G. Sosinsky for their Cx26 collaboration; J. L. Ingraham, J. Houghton, and C. Alston for comments and editing; and C. Alexander for technical assistance. Y-H.H., and J-S.J., were fellows of the Molecular Basis of Disease Program, and J.Z. was a fellow of the Brain and Behavior Program, at Georgia State University. The core facility is supported in part by the Georgia Research Alliance. This work was supported in
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