Rapid and Efficient Extraction of Outer Membrane Proteins from Escherichia coli and Establishment of the Two-Dimensional Electrophoresis Maps

The outer membrane proteins(OMPs) are important part in the Gram-negative bacteria,which are responsible for mass transport,signal recognition and cell attachment.Extraction of outer membrane proteins is the initial step for their further investigation.The traditional extraction methods,however,are time-consuming,complex and difficult to differentiate the outer membrane proteins from the cytoplasm membrane proteins.In this paper,we reported a rapid and efficient method based on temperature-induced phase separation technique to extract selectively the outer membrane proteins from the unbroken Escherichia coli cells.By systematic experimental essaying on the effects of Triton X-114 concentrations and the treatment time on the extraction ofOMPs,we found that 8 % of Triton X-114 treating for 3 hours was the optimal extraction condition.The extracted samples were separated by two-dimensional electrophoresis(2-DE).The 2-DE results indicated that lysis buffer containing low concentration Tris played a key role for the success of 2-D,and that CHAPS combined with ASB-14 orNP-40 could increase the outer membrane proteins solubilization and decrease the isoelectric focusing time.Thus,an efficient 2-D separation system for Escherichia coli outer membrane proteins was established.