Comparison of Techniques for Isolation of the Outer Membrane Proteins of Haemophilus Influenzae Type b

摘要

We compared several rapid techniques used for extraction of outer membrane proteins from gram-negative enteric bacteria to Haemophilus influenzae type b. After lysis of cells with a French press the inner and outer membranes were separated by isopycnic centrifugation. Each membrane was identified by density, morphology, enzymatic activity, and susceptibility to solid-phase iodination of intact cells. By sodium docecyl sulfate-polyacrylamide gel electrophoresis, we identified 10 polypeptides which were enriched in the outer membrane band compared to the inner membrane band. Using these proteins, we compared the polypeptide pattern of outer membranes with that obtained by 1) selective solubilization with sodium dodecyl- -D-maltoside, octyl- -D Glucopyranoside, Triton X-100, sodium or cholamidopropyl dimethylaminopropanesultonate; 2) extraction with chaotropic agent and heat; and 3) differential centrifugation of vesicles shed during transition from log growth phase to stationary growth phase. There were definable differences between the polypeptide pattern of membranes obtained with each rapid technique compared to the polypeptide pattern of isolated outer membranes.