Hahn R, Schulz P M, Schaupp C, Jungbauer A
Institute of Applied Microbiology, University of Agriculture, Forestry and Biotechnology, Vienna, Austria.
J Chromatogr A. 1998 Feb 6;795(2):277-87. doi: 10.1016/s0021-9673(97)01030-3.
Bovine whey proteins have potential applications in veterinary medicine, food industry and as supplements for cell culture media. A fractionation scheme for the economically interesting proteins, such as IgG, lactoferrin and lactoperoxidase, based on cation exchangers was the goal of our investigations. A chromatographic process was developed where alpha-lactalbumin passes through the column and separation of the desired proteins is achieved. Four different cation-exchange media (S-HyperD-F, S-Sepharose FF, Fractogel EMD SO3- 650 (S) and Macro-Prep High S Support) were compared in regard to their dynamic binding capacity for IgG and their different elution behaviours when sequential step gradients with NaCl buffers were applied. Peak fractions were analyzed by size-exclusion chromatography and sodium dodecyl sulphate-polyacrylamide gel electrophoresis. Lactoperoxidase activity was monitored by the oxidation of o-phenylenediamine. In order to explain the different resolution behaviours, isocratic runs with pure standards of whey proteins were performed. The k' values were calculated and plotted against salt concentration. Fractogel EMD had the highest binding capacity for IgG, 3.7 mg/ml gel at a linear flow-rate of 100 cm/h, but the resolution was low compared to that with the other three media. S-Hyper D and S-Sepharose FF showed lower capacities, 3.3 and 3.2 mg/ml gel, respectively, but exhibited better protein resolution. These effects could be partially explained by the k' versus salt concentration plots. The binding capacity of Macro-Prep S was considerably lower compared to that of the other resins investigated because its selectivity for whey proteins was completely different. S-Sepharose FF and S-Hyper D combine relatively high dynamic capacity for IgG and good resolution. Compared to studies with standard proteins, such as 100 mg/ml bovine serum albumin for S-Hyper D, their binding capacities were very low. Even after removal of low-molecular-mass compounds, the capacity could not be improved significantly. The running conditions (low pH) were responsible for the low protein binding capacity, since low-molecular-mass compounds in the feed do not compete with the adsorption of whey protein. The dynamic capacity did not decrease to a large extent within the range of flow-rates (100-600 cm/h) investigated. The dynamic capacity of HyperD and Fractogel was at least five times higher when pure bovine IgG was used for determination. In conclusion, S-Sepharose FF, S-Hyper D-F and Fractogel EMD SO3- 650 (S) are considered as successful candidates for the large-scale purification of bovine whey proteins.
牛乳清蛋白在兽医学、食品工业以及作为细胞培养基补充剂方面具有潜在应用。基于阳离子交换剂对经济上有价值的蛋白质（如免疫球蛋白G、乳铁蛋白和乳过氧化物酶）进行分级分离是我们研究的目标。我们开发了一种色谱方法，在此方法中α-乳白蛋白通过柱子，从而实现所需蛋白质的分离。比较了四种不同的阳离子交换介质（S-HyperD-F、S-Sepharose FF、Fractogel EMD SO3-650(S)和Macro-Prep High S Support）对免疫球蛋白G的动态结合能力以及在应用氯化钠缓冲液进行连续梯度洗脱时它们不同的洗脱行为。通过尺寸排阻色谱法和十二烷基硫酸钠-聚丙烯酰胺凝胶电泳对峰级分进行分析。通过邻苯二胺的氧化监测乳过氧化物酶活性。为了解释不同的分离行为，用乳清蛋白纯标准品进行了等度洗脱实验。计算了k'值并绘制了其与盐浓度的关系图。Fractogel EMD对免疫球蛋白G的结合能力最高，在100 cm/h的线性流速下为3.7 mg/ml凝胶，但与其他三种介质相比分辨率较低。S-Hyper D和S-Sepharose FF的结合能力较低，分别为3.3和3.2 mg/ml凝胶，但蛋白质分辨率较好。这些效应可以通过k'与盐浓度的关系图部分解释。与所研究的其他树脂相比，Macro-Prep S的结合能力相当低，因为它对乳清蛋白的选择性完全不同。S-Sepharose FF和S-Hyper D对免疫球蛋白G具有相对较高的动态容量和良好的分辨率。与用标准蛋白质（如用于S-Hyper D的100 mg/ml牛血清白蛋白）的研究相比，它们的结合能力非常低。即使去除低分子量化合物后，容量也无法显著提高。运行条件（低pH）导致蛋白质结合能力低，因为进料中的低分子量化合物不与乳清蛋白的吸附竞争。在所研究的流速范围（100 - 600 cm/h）内，动态容量没有大幅下降。当使用纯牛免疫球蛋白G进行测定时，HyperD和Fractogel的动态容量至少高出五倍。总之，S-Sepharose FF、S-Hyper D-F和Fractogel EMD SO3-650(S)被认为是大规模纯化牛乳清蛋白的成功候选介质。
