[Purification of outer membrane proteins in Pseudomonas aeruginosa by high performance ion-exchange liquid chromatography]

Abstract

Pseudomonas aeruginosa PAO1 was used in this study. Isolation of outer membrane was accomplished by treating the cell envelope with EDTA and lysozyme, followed by centrifugation. The outer membrane (10 mg of protein) was mixed with 34 mmol/L octyl beta-glucoside-5 mmol/L EDTA-10 mmol/L Tris-HCl (pH 8.0) and subjected to supersonic oscillation for 2 min. The centrifuged supernatant (100 kgf for 30 min at 20 degrees C) was applied onto a DEAE ion-exchange high performance liquid chromatographic column (TSK gel-DEAE-5PW column, 0.75 cm x 7.5 cm i.d.) that was equilibrated with a solution of 10 mmol/L Tris-HCl buffer (pH 8.0) containing 2.5 mmol/L beta-C12E8 and 1 mmol/L EDTA. The column was washed with the same solution and eluted with a linear gradient of 0-0.5 mol/L NaCl in the same solution and fractions A, B, C were collected. Proteins in these fractions were analyzed by SDS-polyacrylamide gel electrophoresis and quantified by the method of Lowry et al. Protein E(Mr 43,000), G(Mr 25,000) and H (Mr 19,000) flowed through the column without adsorption in fraction A. Protein C(Mr 70,000), D(Mr 46,000) and a small amount of F (Mr 34,000) were eluted in fraction B. Fraction A was concentrated with ultrafiltration and applied again onto a DEAE ion-exchange HPLC column equilibrated with 10 mmol/L Tris-HCl buffer, pH 8.0, containing 34 mmol/L beta-C12E8 and 1 mmol/L MgCl2. Fraction B was subjected to DEAE ion-exchange HPLC column in the presence of EDTA. This fraction was then applied onto a DEAE ion-exchange HPLC column equilibrated with 10 mmol/L Tris-HCl buffer, pH 8.0, containing 34 mmol/L octylglucoside and 1 mmol/L EDTA. By these procedures protein C, D and E were purified to apparent homogeneity as judged by SDS-PAGE. In this work, we purified the outer membrane proteins of Pseudomonas aeruginosa, and used a new technique selectively solubilizing the cytoplasmic membrane with sodium lauryl sarcosinate for isolating the outer membrane proteins of Pseudomonas aeruginosa because of its relative simplicity.