Probiodrug AG, Weinbergweg, Halle, Germany.
Biol Chem. 2011 Jul;392(7):665-77. doi: 10.1515/BC.2011.068.
Abstract In the present studies we resolved the post-translational microheterogeneity of purified porcine dipeptidyl peptidase IV (DP 4) from kidney cortex. Applying SDS-homogeneous DP 4 onto an analytical agarose isoelectric focusing (IEF) gel, pH 4-6, activity staining resulted in at least 17 isoforms between pH 4.8-6.0. These could be separated into fractions with only two to six isoforms by means of preparative liquid-phase IEF, using a Rotofor cell. Starting off with three parallel Rotofor runs under the same conditions at pH 5-6, the fractions were pooled according to the specific activity of DP 4, pH and analytical IEF profile, and further refractionated without any additional ampholytes. Since excessive dilution of ampholytes and proteins was kept to the minimum, a second refractionation step could be introduced, resulting in pH gradients between 0.022 and 0.028 pH increments per fraction. By performing two consecutive refractionation steps, the high resolution necessary for the separation of DP 4 isoforms could be achieved. This represents an alternative method if isolation of isoforms with similar pI's results in precipitation and denaturation in presence of a narrow pH range. Furthermore, it demonstrates that preparative IEF is a powerful tool to resolve post-translational microheterogeneity of a purified protein required for crystallization processing.
摘要 在本研究中，我们解决了从猪肾皮质中纯化的二肽基肽酶 IV（DP4）的翻译后微观异质性。将 SDS 均匀 DP4 施加到分析性琼脂糖等电聚焦（IEF）凝胶（pH4-6）上，活性染色导致 pH4.8-6.0 之间至少存在 17 种同工型。这些同工型可以通过使用 Rotofor 细胞的制备液相IEF 分离为仅具有两种到六种同工型的分数。从三个平行的 Rotofor 运行开始，在 pH5-6 下进行相同条件下运行，根据 DP4 的比活性、pH 值和分析 IEF 图谱将分数汇集在一起，并在没有任何额外的两性电解质的情况下进一步重新分级。由于保持了对两性电解质和蛋白质的过度稀释最小化，因此可以引入第二个重新分级步骤，导致每个分数之间的 pH 梯度为 0.022 至 0.028 pH 增量。通过执行两个连续的重新分级步骤，可以实现 DP4 同工型分离所需的高分辨率。如果在存在窄 pH 范围的情况下，同 pI 的同工型的分离导致沉淀和变性，则这是一种替代方法。此外，它表明制备性IEF 是一种强大的工具，可用于解决需要结晶处理的纯化蛋白的翻译后微观异质性。
