Rapid And Efficient Extraction Of Outer Membrane Proteins From Acidithiobacillus Ferrooxidans And Establishment Of The Two-dimensional Electrophoresis Maps

Acidithiobacillus ferrooxidans (A. ferrooxidans) is a chemolithoautotrophic, Gram-negative bacterium. The bacterium can get energy for growth from the oxidation of ferrous to ferric iron and elemental sulfur or reduced inorganic sulfur compounds to sulfate using oxygen as electron acceptor. The outer membrane proteins (OMPs) are important part in the Gram-Negative bacterium, which are responsible for mass transport, signal recognition and cell attachment. Extraction of OMPs is the initial step for their further investigation. The traditional extraction methods, however, are time-consuming, complexity and difficult to differentiate the OMPs from the cytoplasm membrane proteins.In this paper, we report a rapid and efficient method based on temperature-induced phase separation technique to extract selectively the OMPs. By systematic experimental assay effects of Triton X-114 concentrations and the treated time on the extraction efficiency of OMPs, we found that 8% of Triton X-114 treating for 3 hours is the optimal extraction condition. The extracted samples were separated by two-dimensional electrophoresis (2-DE). The 2-DE results indicate that lysis buffer containing low concentration Tris is a key element for the success of 2-DE. It can increase the OMPs solubilization and decrease the isoelectric focusing time when CHAPS was combined with ASB-14 or NP-40. A good separation 2-DE system for Escherichia coli OMPs was established.Then we used the method to extract the OMPs of A. ferrooxidans, and established two - dimensional electrophoresis maps of OMPs of A. ferrooxidans. These work pave a way for the further research of OMPs of A. ferrooxidans.