Measuring protein interactions by microchip self-interaction chromatography
Measuring protein interactions by microchip self-interaction chromatography
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DOI：
10.1021/bp025788z
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发表时间：
2003-05-01
影响因子：
2.9
通讯作者：
Henry, CS
中科院分区：
文献类型：
--
作者：
García, CD;Hadley, DJ;Henry, CS
蛋白质的自我相互作用在聚集和结晶现象中起着至关重要的作用。导致蛋白质聚集状态改变的溶液条件，无论是无定形的还是结晶的，主要是通过使用大量溶液的试错筛选来发现的。自相互作用层析有可能为高通量筛选提供一种定量测定蛋白质自身相互作用的方法。本文介绍了一种以溶菌酶为模型蛋白质的低压自作用层析芯片分离系统的结构和特性。分析了流动相组成、蛋白质进样量、流速和固定相修饰对保留时间的影响。将作为结晶剂浓度函数的容量因数(k‘)与先前公布的通过静态光散射获得的渗透第二维里系数(B-22)值进行比较，表明该芯片能够准确地确定蛋白质-蛋白质相互作用。与目前使用的评估蛋白质相互作用的方法相比，蛋白质消耗减少500倍，以及使用传统仪器和自动化的可能性是一些优势。
The self-interaction of proteins is of paramount importance in aggregation and crystallization phenomena. Solution conditions leading to a change in the state of aggregation of a protein, whether amorphous or crystalline, have mainly been discovered by the use of trial and error screening of large numbers of solutions. Self-interaction chromatography has the potential to provide a quantitative method for determination of protein self-interactions amenable to high-throughput screening. This paper describes the construction and characterization of a microchip separation system for low-pressure self-interaction chromatography using lysozyme as a model protein. The retention time was analyzed as a function of mobile-phase composition, amount of protein injected, flow rate, and stationary-phase modification. The capacity factors (k') as a function of crystallizing agent concentration are compared with previously published values for the osmotic second virial coefficient (B-22) obtained by static light scattering, showing the ability of the chip to accurately determine protein-protein interactions. A 500-fold reduction in protein consumption and the possibility of using conventional instrumentation and automation are some of the advantages over currently used methodologies for evaluating protein-protein interactions.