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Miniaturized parallel screens to identify chromatographic steps required for recombinant protein purification

Nature Protocols volume 5, pages 408–417 (2010)Cite this article

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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 ∼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.

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Figure 1
Figure 2: Analysis of fractions eluted during the miniaturized screening using sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE).
Figure 3: SDS-PAGE analysis of fractions eluted from the second screening step for the purification of α-amylase from cell-free culture broth.

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Authors and Affiliations

  1. Department of Chemical Engineering, ECG 202, Arizona State University, Tempe, Arizona, USA

    Kaushal Rege

  2. A Danisco Division, Process Engineering and Technology, Genencor, Palo Alto, California, USA

    Meng Heng

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  1. Kaushal Rege
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  2. Meng Heng
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Contributions

K.R. and M.H. designed the work, K.R. carried out experiments, and K.R. and M.H. analyzed the data and wrote the manuscript.

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Correspondence to Kaushal Rege.

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Rege, K., Heng, M. Miniaturized parallel screens to identify chromatographic steps required for recombinant protein purification. Nat Protoc 5, 408–417 (2010). https://doi.org/10.1038/nprot.2009.149

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