Characterization of A Cyanobacterial Outer Membrane Protein: an E. Coli Tolc Homologue from Synechocystis Sp Pcc 6803

E. coli TolC (tolerance to colicins) represents an interesting class of outer membrane (OM) proteins, as it has an α-helical periplasmic tunnel and β-barrel membrane region, providing a conduit for export of metabolites and xenobiotics from cell interior to exterior, and import of colicin E1 (1). A TolC homologue, Slr1270 from Synechocystis 6803, cloned and expressed in E. coli, has > 40% similarity and ∼16% identity to E. coli and Pseudomonas counterparts, and has a similar domain organization. Homology modeling using Pseudomonas OprM as template modeled 93% of Slr1270 sequence. The 1581bp slr1270 gene was cloned and overexpressed in E. coli. Protein from inclusion bodies, refolded through step-wise dialysis showed major bands at ∼55kDa and >150 kDa on SDS-PAGE corresponding to the monomer and trimer respectively and ∼300 kDa on CN-PAGE. Purified protein displays a far-UV CD spectrum characteristic of E. coli TolC with > 50% α-helix, and formed channels in planar lipid bilayers with a characteristic single channel conductance of ∼50 pS in 0.1M NaCl. The intact protein mass spectrum (LC-MS) with a major peak at 54,489 probably represents a mixture of two species, the TolC product with 1-40 removed and an intact 6-His tag (calculated mass 54,457.1 Da), and a product with 1-38 removed and a 5-His tag (calculated mass 54,490.2 Da) after a single carboxypeptidase event. The small peak at 54,638 Da probably corresponds to TolC product with 1-38 removed and an intact 6-His tag (calculated mass 54,627.3 Da). Peptides 39-76 and 41-76 were recovered from trypsin digests confirming N-terminal heterogeneity. (1) Zakharov, S. D. et al. 2012. Pathways of Colicin Import: Utilization of BtuB, OmpF Porin, and the TolC Drug Export Protein, Biochem. Soc. Trans., 40, 1463-1468.