High-throughput protein purification under denaturating conditions by the use of cation exchange chromatography

Abstract

A high-throughput protein purification strategy using the polycationic Zbasic tag has been developed. In order for the strategy to be useful both for soluble and less soluble proteins, a denaturating agent, urea, was used in all purification steps. First, four target proteins were genetically fused to the purification tag, Zbasic. These protein constructs were purified by cation exchange chromatography and eluted using a salt gradient. From the data achieved, a purification strategy was planned including stepwise elution to enable parallel protein purification using a laboratory robot. A protocol that includes all steps, equilibration of the chromatography resin, load of sample, wash, and elution, all without any manual handling steps, was handled by the laboratory robot. The program allows automated purification giving milligram amounts of pure recombinant protein of up to 60 cell lysates. In this study 22 different protein constructs, with different characteristics regarding p/ and solubility, were successfully purified by the laboratory robot. The data show that Zbasic can be used as a general purification tag also under denaturating conditions. Moreover, the strategy enables purification of proteins with different p / and solubility using ion exchange chromatography (IEXC). The procedure is highly reproducible and allows for high protein yield and purity and is therefore a good complement to the commonly used His6-tag. © 2007 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.