Expression and refolding of Omp38 from Burkholderia pseudomallei and Burkholderia thailandensis, and its function as a diffusion porin

摘要

pIn the present paper, we describe cloning and expression of two outer membrane proteins, iBps/iOmp38 (from iBurkholderia pseudomallei/i) and iBth/iOmp38 (from iBurkholderia thailandensis/i) lacking signal peptide sequences, using the pET23d(+) expression vector and iEscherichia coli/i host strain Origami(DE3). The 38 kDa proteins, expressed as insoluble inclusion bodies, were purified, solubilized in 8 M urea, and then subjected to refolding experiments. As seen on SDS/PAGE, the 38 kDa band completely migrated to ～110 kDa when the purified monomeric proteins were refolded in a buffer system containing 10% (w/v) Zwittergent? 3-14, together with a subsequent heating to 95 °C for 5 min. CD spectroscopy revealed that the 110 kDa proteins contained a predominant β-sheet structure, which corresponded completely to the structure of the Omp38 proteins isolated from iB. pseudomallei/i and iB. thailandensis/i. Immunoblot analysis using anti-iBps/iOmp38 polyclonal antibodies and peptide mass analysis by MALDI–TOF (matrix-assisted laser-desorption ionization–time-of-flight) MS confirmed that the expressed proteins were iBps/iOmp38 and iBth/iOmp38. The anti-iBps/iOmp38 antibodies considerably exhibited the inhibitory effects on the permeation of small sugars through the Omp38-reconstituted liposomes. A linear relation between relative permeability rates and iM/isubr/sub of neutral sugars and charged antibiotics suggested strongly that the iin vitro/i re-assembled Omp38 functioned fully as a diffusion porin./p