Automated protein purification using His‐tagged affinity chromatography with the Profinia protein purification system.

Abstract

Immobilized Metal Affinity Chromatography (IMAC) has become one of the most popular technologies for purifying recombinant proteins. Using high-capacity IMAC resins, His-tagged proteins can be obtained at high yields from crude extracts without prior removal of other cellular components. An appealing feature of IMAC is that a standardized protocol can be applied to a diverse range of His-tagged proteins, which allows efficient purification for proteomics research and protein structural studies. Difficulties commonly encountered in His-tagged protein expression and purification includes low expression level and low solubility of over expressed proteins. The Profinia protein purification system, an automated low pressure chromatography system, has been used to enrich both poorly expressed and low solubility proteins, resulting in up to 7.5 mg of target protein that is more than 90 % pure from 500 ml of bacterial culture. Profinia comes preprogrammed with standard IMAC methods, but is also customizable for optimizing purification protocols. Optimization of IMAC purification parameters for a challenging protein, and representative yield and purity data for multiple proteins will be presented.