A novel two-dimensional protein separation method and its applications

A novel two-dimensional procedure for separating proteins according to their isoelectric points under non-denaturing conditions employing blotting membranes is described. Neither ampholytes nor preformed pH gradient strips are required for this new two-dimensional High Performance Liquid Electrophoresis (2D-HPLE) procedure. Proteins are separated on blot membranes such as PVDF or nylons in two dimensions using water-miscible organic solvent buffers. By carrying out 2D-HPLE under non-denaturing conditions both free and complexed proteins (e.g. protein-protein complexes) are separated. Each of the two dimensions requires only about 0.3 kVh as compared to 30 or more kVh for the regular IEF using preformed pH gradients. Thus, the entire 2D-HPLE process (including protein detection on membranes) can be completed in about 40 min vs. about 2 days for the regular 2D-PAGE. Only about 5 μg of proteins are required to perform the 2D-HPLE. Hydrophobic proteins are separated into exceptionally clean and well resolved spots without the diffusion and streaking normally observed with regular 2D-PAGE. Hydrophilic proteins are also similarly resolved. By separating proteins directly on PVDF membranes, they can be directly used for immunoprobing (Western blot) and mass spectrometry. Because the 2D-HPLE process is carried out in the absence of denaturing agents such as urea and sodium dodecyl sulfate, the retention of the activities of enzymes that were tested was observed. Separation of proteins can also be carried out one-dimensionally to analyze protein compositions based on charge. This high-speed, high-resolution 2-D separation method possesses many advantages not found in the conventional 2D-SDS-PAGE and will be very useful in the fields of both “expression proteomics” and “functional proteomics”.; Using HPLE a new broad spectrum protease inhibitor and its endogenous protease were purified and characterized from the entomopathogenic bacterium Photorhabdus luminescens. The complete amino acid sequence for the protease inhibitor was determined and interestingly there appears to be twenty amino acids prior to the N-terminal sequence which indicates the need for the protease inhibitor to be activated in order to become active. The implications and applications of this newly discovered protease inhibitor are discussed.