title: Miniaturized parallel screens to identify chromatographic steps required for recombinant protein purification.
abstract: Methods development in chromatography is a time-consuming, trial-and-error process that requires laborious experimentation. We describe a high-throughput screening (HTS) protocol for the rapid identification of chromatographic steps for protein purification from cell-free expression broths. Broths containing the protein are loaded on different chromatographic resins aliquotted in membrane-bottomed microtiter plates. Serial step elution of protein from resins results in fraction collection in 96-well plates. Choice of the optimal chromatographic operating conditions is based on protein purity in eluted fractions, determined using SDS-PAGE analysis or similar analytical techniques. The screening procedure is then repeated in order to identify the subsequent chromatographic steps, ultimately leading to high purities of the protein. The protocol takes approximately 24 h in order to determine the required sequence of chromatographic steps. The use of a miniaturized screen facilitates screening of a range of media and operating conditions (i.e., pH, salt concentration, and so on.) in parallel and is a novel approach to chromatographic methods development.
authors.affiliation: Department of Chemical Engineering, Arizona State University, Tempe, Arizona, USA. Kaushal.Rege@asu.edu
authors.forename: Kaushal
authors.initials: K
authors.lastname: Rege
authors.forename: Meng
authors.initials: M
authors.lastname: Heng
mesh_terms: D001709:Biotechnology; D002845:Chromatography / Q000379:methods*; D002846:Chromatography, Affinity / Q000379:methods; D002852:Chromatography, Ion Exchange / Q000379:methods; D007475:Ion Exchange Resins; D008904:Miniaturization / Q000379:methods; D011994:Recombinant Proteins / Q000302:isolation & purification*; D000516:alpha-Amylases / Q000302:isolation & purification
chemical_list: D007475:Ion Exchange Resins; D011994:Recombinant Proteins; D000516:alpha-Amylases