Protein Expression and Purification ( IF 1.6 ) Pub Date : 2020-07-16 , DOI: 10.1016/j.pep.2020.105698 Sara Kanje 1 , Henric Enstedt 1 , Melanie Dannemeyer 1 , Mathias Uhlén 1 , Sophia Hober 1 , Hanna Tegel 1
The Human Secretome Project aims to produce and purify all human secreted proteins as full-length. In order to enable this, a robust, gentle and effective purification process is needed, where multiple proteins can be purified in parallel. For this reason, a purification system based on a Protein C-tag and the HPC4 antibody with high affinity to the tag was chosen for purification. The strong binding between the tag and the antibody is specific and calcium-dependent, which allows for mild elution with EDTA. Presented here is a study comparing different protein purification base matrices coupled with the HPC4 antibody, aiming to increase the yield of purified protein and reduce the time for purification. Among the different tested matrices, Capto XP showed a high coupling degree and increased the amount of eluted protein as compared to the control matrix. By moving from batch incubation to direct sample loading and by performing the purification on the ÄKTAxpress, an automated protein purification process and a high reduction of hands-on sample handling was achieved. This new method also integrates the desalting step in the purification process, and the time for purification and analysis of each sample was decreased from five to three days. Moreover, a new mild method for matrix regeneration was developed using 50 mM EDTA pH 7.5 instead of 0.1 M glycine pH 2. This method was proven to be efficient for regeneration while maintaining the column binding performance even after nine rounds of regeneration.
中文翻译:
使用钙依赖性设置改进高通量蛋白质纯化工艺。
Human Secretome Project旨在生产和纯化所有人类分泌的全长蛋白质。为了做到这一点,需要一种健壮,温和且有效的纯化方法,其中可以并行纯化多种蛋白质。因此,选择基于蛋白质C标签和对标签具有高亲和力的HPC4抗体的纯化系统进行纯化。标签和抗体之间的强结合是特异性的和钙依赖性的,从而允许用EDTA温和洗脱。本文介绍了一项比较不同蛋白质纯化基础基质与HPC4抗体的研究,目的是提高纯化蛋白质的收率并减少纯化时间。在不同的测试矩阵中,与对照基质相比,Capto XP显示出高偶联度并增加了洗脱蛋白的量。通过从分批培养转变为直接上样,并在ÄKTAxpress上进行纯化,可以实现自动化的蛋白质纯化过程并大大减少了动手操作样品的过程。这种新方法还将脱盐步骤集成到了纯化过程中,每个样品的纯化和分析时间从五天减少到三天。此外,使用50 mM EDTA pH 7.5代替0.1 M甘氨酸pH 2开发了一种新的温和的基质再生方法,该方法被证明是高效的再生方法,即使经过9轮再生也可以保持色谱柱结合性能。通过从分批培养转变为直接上样,并在ÄKTAxpress上进行纯化,可以实现自动化的蛋白质纯化过程并大大减少了动手操作样品的过程。这种新方法还将脱盐步骤集成到了纯化过程中,每个样品的纯化和分析时间从五天减少到三天。此外,使用50 mM EDTA pH 7.5代替0.1 M甘氨酸pH 2开发了一种新的温和的基质再生方法,该方法被证明是高效的再生方法,即使经过9轮再生也可以保持色谱柱结合性能。通过从分批培养转变为直接上样,并在ÄKTAxpress上进行纯化,可以实现自动化的蛋白质纯化过程并大大减少了动手操作样品的过程。这种新方法还将脱盐步骤集成到了纯化过程中,每个样品的纯化和分析时间从五天减少到三天。此外,使用50 mM EDTA pH 7.5代替0.1 M甘氨酸pH 2开发了一种新的温和的基质再生方法,该方法被证明是高效的再生方法,即使经过9轮再生也可以保持色谱柱结合性能。这种新方法还将脱盐步骤集成到了纯化过程中,每个样品的纯化和分析时间从五天减少到三天。此外,使用50 mM EDTA pH 7.5代替0.1 M甘氨酸pH 2开发了一种新的温和的基质再生方法,该方法被证明是高效的再生方法,即使经过9轮再生也可以保持色谱柱结合性能。这种新方法还将脱盐步骤集成到了纯化过程中,每个样品的纯化和分析时间从五天减少到三天。此外,使用50 mM EDTA pH 7.5代替0.1 M甘氨酸pH 2开发了一种新的温和的基质再生方法,该方法被证明是高效的再生方法,即使经过9轮再生也可以保持色谱柱结合性能。