Pflegerl K, Podgornik A, Berger E, Jungbauer A
Institute of Applied Microbiology, University of Agricultural Sciences, Muthgasse 18, Vienna, Austria.
Biotechnol Bioeng. 2002 Sep 30;79(7):733-40. doi: 10.1002/bit.10330.
Screening of peptide ligands for affinity chromatography usually involves incubation with the target protein in a batch system. In an additional step, peptides with fast binding kinetics have to be selected in respect to satisfactory performance under flow conditions on a support ensuring optimal three-dimensional presentation of the peptide. We have developed a rapid screening system based on peptide synthesis and screening on CIM((R)) disks. The disk size was minimized to fit into microplates usually applied for solid-phase extraction. In combination with a vacuum manifold, semi-automated peptide synthesis and screening for binding to a target protein under simulated chromatography conditions are possible. Various analytical methods can be applied for parallel and automated determination of the quantity, integrity, or activity of the target protein in the flow through or bound to the affinity support. This system also allows parallel screening for suitable chromatographic conditions like running buffer, washing, and elution conditions.
用于亲和色谱的肽配体筛选通常涉及在批量系统中与目标蛋白孵育。在额外的步骤中,必须选择具有快速结合动力学的肽,以确保在流动条件下在支持物上具有令人满意的性能,从而保证肽的最佳三维呈现。我们开发了一种基于肽合成并在CIM((R))盘上进行筛选的快速筛选系统。盘的尺寸被最小化,以适合通常用于固相萃取的微孔板。与真空歧管相结合,可以在模拟色谱条件下进行半自动肽合成以及筛选与目标蛋白的结合情况。可以应用各种分析方法,并行且自动地测定流过亲和支持物或结合在亲和支持物上的目标蛋白的量、完整性或活性。该系统还允许并行筛选合适的色谱条件,如运行缓冲液、洗涤和洗脱条件。