Channel forming outer membrane porin protein in halophile: Expressed as a soluble form in Escherichia coli

We have previously found that the N-terminal sequence of the outer membrane protein from moderate halophile is similar to the sequence of the well-known pore forming porin proteins from other Gram-negative bacteria. This highly expressed outer membrane protein was purified from Halomonas sp. 40 and reconstituted into liposome. It showed a permeability activity in the liposome swelling assay. Based on the N-terminal and internal amino acid sequences of this major outer membrane, we have cloned here the porin gene, hopP (halophilic outer membrane protein), from Halomonas sp. 40. The hopP gene encodes the porin precursor comprising 366 amino acid residues that include a 21 amino acid signal peptide. Mature porin (345 amino acids, 37,611 Da) is a highly acidic protein, just as is so for many halophilic proteins and was soluble when expressed in Escherichia coli with N-terminal His-tag. Purified recombinant His-porin was soluble even after heat-treatment at 95 degrees C for 5 min in the absence of salt. Circular dichroism analysis of His-porin showed conversion into a beta-sheet rich structure by the addition of NaCl at 0.9-2.7 M. (C) 2012 Elsevier B.V. All rights reserved.