Jaruwan SiritapetaweeHeino PrinzChartchai KrittanaiWipa SugintaSuranaree University of TechnologyMax Planck Institut fur molekulare PhysiologieMahidol UniversityNational Synchrotron Research Center, Thailand2018-07-242018-07-242004-12-15Biochemical Journal. Vol.384, No.3 (2004), 609-617026460212-s2.0-10944239453https://repository.li.mahidol.ac.th/handle/20.500.14594/21110In the present paper, we describe cloning and expression of two outer membrane proteins, BpsOmp38 (from Burkholderia pseudomallei) and BthOmp38 (from Burkholderia thailandensis) lacking signal peptide sequences, using the pET23d(+) expression vector and Escherichia coli 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 B. pseudomallei and B. thailandensis. Immunoblot analysis using anti-BpsOmp38 polyclonal antibodies and peptide mass analysis by MALDI-TOF (matrix-assisted laser-desorption ionization-time-of-flight) MS confirmed that the expressed proteins were BpsOmp38 and BthOmp38. The anti-BpsOmp38 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 Mr of neutral sugars and charged antibiotics suggested strongly that the in vitro re-assembled Omp38 functioned fully as a diffusion porin.Mahidol UniversityBiochemistry, Genetics and Molecular BiologyArticleSCOPUS10.1042/BJ20041102