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

Abstract

In 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 approximately 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 degrees C for 5 min. CD spectroscopy revealed that the 110 kDa proteins contained a predominant beta-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 M(r) of neutral sugars and charged antibiotics suggested strongly that the in vitro re-assembled Omp38 functioned fully as a diffusion porin.

Antibiotic resistance in Burkholderia species

“…Little is known about porins and their possible roles in B. pseudomallei drug resistance. In vitro studies with B. pseudomallei Omp38 in reconstituted liposomes indicated that this protein functions as a diffusion porin for neutral sugars and charged antibiotics (Siritapetawee et al, 2004). Subsequent black lipid membrane reconstitution studies, liposome swelling assays and expression in a porin-deficient E. coli strain confirmed translocation of antibiotics through Omp38 and suggested a possible contribution of this porin in B. pseudomallei ceftazidime and carbapenem resistance (Aunkham et al, 2014; Suginta et al, 2011).…”

Crystal structures of theBurkholderia multivoranshopanoid transporter HpnN

“…Most of these species contain a modified lipopolysaccharide, which results in polymyxin resistance (13). In addition, the permeability of the major outer membrane porin channel Omp38 appears to be low for antibiotics (14). The presence of a large number of multidrug efflux pumps, belonging to the resistance-nodulation-cell division (RND) superfamily, also plays a major role in the intrinsic resistance to a variety of antimicrobials.…”

Porin Involvement in Cephalosporin and Carbapenem Resistance of Burkholderia pseudomallei

“…As shown in Fig. 2, the purified protein, which migrated as a band of 42 kDa on SDS-PAGE, was equivalent to Bps Omp38 isolated from the outer membrane of native Bps [22]. After purification, 1–2 mg of recombinant Bps Omp38 per litre of bacterial culture was obtained; this was 5–10 times higher than was obtained using the unfolding/refolding protocol [22].…”

“…2, the purified protein, which migrated as a band of 42 kDa on SDS-PAGE, was equivalent to Bps Omp38 isolated from the outer membrane of native Bps [22]. After purification, 1–2 mg of recombinant Bps Omp38 per litre of bacterial culture was obtained; this was 5–10 times higher than was obtained using the unfolding/refolding protocol [22]. Although in the E. coli BL21 (Omp8) Rosetta strain the genes encoding OmpF, OmpC, OmpA and LamB are disrupted [25], our previous study showed that this bacterium could still express a significant amount of endogenous OmpN, as a major contaminant [38].…”