title: Channel forming outer membrane porin protein in halophile: expressed as a soluble form in Escherichia coli.
abstract: 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 °C for 5 min in the absence of salt. Circular dichroism analysis of His-porin showed conversion into a β-sheet rich structure by the addition of NaCl at 0.9-2.7 M.
authors.affiliation: Applied and Molecular Microbiology, Faculty of Agriculture, Kagoshima University, 1-21-24 Korimoto, Kagoshima 890-0065, Japan.
authors.forename: Hiroko
authors.initials: H
authors.lastname: Tokunaga
authors.forename: Masafumi
authors.initials: M
authors.lastname: Furukawa
authors.forename: Tsutomu
authors.initials: T
authors.lastname: Arakawa
authors.forename: Masao
authors.initials: M
authors.lastname: Tokunaga
mesh_terms: D001425:Bacterial Outer Membrane Proteins / Q000378:metabolism*; D001483:Base Sequence; D002942:Circular Dichroism; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism*; D020632:Halomonas / Q000378:metabolism*; D006639:Histidine / Q000378:metabolism; D006358:Hot Temperature; D008081:Liposomes / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000378:metabolism; D018272:Porins / Q000378:metabolism*; D021381:Protein Transport; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012995:Solubility; D055672:Static Electricity; D013347:Subcellular Fractions / Q000378:metabolism
chemical_list: D001425:Bacterial Outer Membrane Proteins; C471213:His-His-His-His-His-His; D008081:Liposomes; D009842:Oligopeptides; D018272:Porins; D011993:Recombinant Fusion Proteins; D006639:Histidine