Micropreparative Purification of Protein Isoforms by Dynamic Focusing

该奖项支持继续开发一种新型的电泳仪，用于分离、浓缩和回收微克量的纯化蛋白质异构体和糖体。基本的电聚焦原理，动态场梯度聚焦[DFGF]，使用计算机控制的电极阵列来建立和实时处理电场梯度。梯度将驱动蛋白质与填充柱中恒定的缓冲液流动，使每种蛋白质移动到柱中的位置，在那里其电泳速度与其层析速度相平衡。由于DFGF的分离能力在很大程度上受到电子控制器保真度的限制，因此高精度电子设备的使用应该会使该仪器的性能优于基于等电聚焦的传统仪器。此外，电场梯度可以在运行期间根据由适当检测器收集的信息进行调整。通过在运行过程中改变电场分布，应该可以定位特定的单个蛋白质并从柱出口顺序捕获它们。然后，可以用标准的分析工具对分离的蛋白质进行分析，以确定氨基酸序列、碳水化合物组成和其他化学性质的差异。作为该项目的一部分，该仪器将使用来自工业和学术实验室的重组蛋白来测试该仪器自动将纯化的蛋白质分离成各种异构体和糖型的能力。许多蛋白质，无论是保留在细胞内还是分泌出来，都已被证明通过添加磷酸残基、糖残基或其他类型的有机或无机小分子在一个或多个位置进行修饰。近年来，对单个蛋白质结构和功能的研究，包括对酶的构象和活性调节的研究，越来越受到翻译后修饰的性质、位置和作用的关注。通常使用等电聚焦结合凝胶或毛细管电泳法可以对给定蛋白质的各种形式进行表征和纯化；然而，通常很难从凝胶或毛细管中分离和回收足够的每一种异构体，以便能够进一步表征蛋白质的天然状态。随着蛋白质研究在未来几年继续扩大，将在该奖项的支持下开发的设备应该会改善蛋白质化学家可用的选择范围。

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.