A comparison of techniques for isolation of the outer membrane proteins of Haemophilus influenzae type b.
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 dodecyl 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-beta-D-maltoside, octyl-beta-D-glucopyranoside, Triton X-100, sodium, or cholamidopropyl dimethylaminopropanesulfonate; (2) extraction with chaotropic agents 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. The polypeptide pattern of lithium extracts and the Triton X-100 insoluble fractions of total membranes most closely approximated the polypeptide pattern of isopycnically isolated outer membranes. Depending on the outer membrane protein sought, one of these rapid techniques can be utilized when a rapid method of outer membrane protein isolation is required.
D001425:Bacterial Outer Membrane Proteins / Q000302:isolation & purification*; D002500:Centrifugation, Isopycnic; D004591:Electrophoresis, Polyacrylamide Gel; D006193:Haemophilus influenzae / Q000032:analysis*; D007455:Iodine; D008854:Microscopy, Electron; D010455:Peptides / Q000032:analysis; D012588:Scintillation Counting; D012995:Solubility
D001425:Bacterial Outer Membrane Proteins; D010455:Peptides; D007455:Iodine