Micropreparative Purification of Protein Isoforms by Dynamic Focusing

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This award supports the continued development of a new type of electrophoresis instrument for isolation, concentration and recovery of microgram amounts of purified protein isoforms and glycoforms in their native state. The underlying electrofocusing principle, dynamic field-gradient focusing [DFGF], uses a computer-controlled array of electrodes to establish and manipulate, in real time, an electric field gradient. The gradient will drive the protein against a constant flow of buffer in a packed column so that each protein moves to a position in the column where its electrophoretic velocity counterbalances its chromatographic velocity. Because the separating power of DFGF is limited largely by the fidelity of the electronic controller, the use of high-precision electronics should allow this instrument to outperform conventional instruments based on the use of isoelectric focusing. Moreover, the electric-field gradient can be adjusted during a run in response to information collected by an appropriate detector. By altering the electric field profile during the run, it should be possible to localize specific individual proteins and capture them sequentially from the column outlet. The isolated proteins can then be analyzed by standard analytical tools to determine differences in amino acid sequence, carbohydrate composition, and other chemical properties. As part of the project, the ability of this instrument to automatically fractionate purified proteins into their various isoforms and glycoforms will be tested using recombinant proteins from industrial and academic laboratories.Many proteins, whether retained in the cell or secreted, have been shown to be modified at one or more sites by the addition of phosphate residues, sugar residues or other types of small organic or inorganic molecules. In recent years, the study of the structure and function of individual proteins, including study of the regulation of enzyme conformation and activity, has been increasingly concerned with the nature, location and effect of post-translational modifications. The various forms of a given protein can be usually characterized and purified using isoelectric focusing combined with gel or capillary electrophoresis; however, it is frequently difficult to separate and recover enough of each isoform from a gel or capillary to be able to further characterize the protein in its native state. The device to be developed with support from this award should improve the range of options available to protein chemists as the study of proteins continues to expand in the coming years.