A blue native-PAGE analysis of membrane protein complexes in Clostridium thermocellum

Abstract

Background: Clostridium thermocellum is a Gram-positive thermophilic anaerobic bacterium with the unusual capacity to convert cellulosic biomass into ethanol and hydrogen. Identification and characterization of protein complexes in C. thermocellum are important toward understanding its metabolism and physiology.

Results: A two dimensional blue native/SDS-PAGE procedure was developed to separate membrane protein complexes of C. thermocellum. Proteins spots were identified by MALDI-TOF/TOF Mass spectrometry. 24 proteins were identified representing 13 distinct protein complexes, including several putative intact complexes. Interestingly, subunits of both the F1-F0-ATP synthase and the V1-V0-ATP synthase were detected in the membrane sample, indicating C. thermocellum may use alternative mechanisms for ATP generation.

Conclusion: Two dimensional blue native/SDS-PAGE was used to detect membrane protein complexes in C. thermocellum. More than a dozen putative protein complexes were identified, revealing the simultaneous expression of two sets of ATP synthase. The protocol developed in this work paves the way for further functional characterization of these protein complexes.

Figure 1

Coomassie blue-stained 2D BN/SDS-PAGE separation of membrane protein complexes of C. thermocellum. Approximately 40 μg of protein was loaded in the first dimensional BN-PAGE lane. Sizes of molecular mass markers are indicated on the top of BN-P|AGE gel and at the left of the SDS gel. The slice of first dimensional BN-PAGE separation gel was placed on top of the second dimensional SDS-PAGE gel and resolved. Protein spots picked for mass spectrometry analysis are marked by arrows and numbered.