Use of proteomics for design of a tailored host cell for highly efficient protein purification |
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Authors: | Zhu Liu Patrick Bartlow Rajaramesh Varakala Robert Beitle Richard Koepsel Mohammad M Ataai |
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Institution: | 1. Department of Chemical and Petroleum Engineering, University of Pittsburgh, 1249 Benedum Hall, 3700 O’Hara St., Pittsburgh, PA 15261, USA;2. Department of Bioengineering, University of Pittsburgh, 306 Center for Biotechnology, 300 Technology Drive, Pittsburgh, PA 15219, USA;3. Department of Chemical Engineering, University of Arkansas, 3202 Bell Engineering Center, Fayetteville, AR 72701, USA |
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Abstract: | After some initial optimization, a downstream process comprised of one or several chromatography steps removes the majority of the host proteins and achieves a reasonable degree of purification. The separation of remaining contaminant proteins from the target protein could become very difficult and costly due to their similar physicochemical properties. In this paper we describe a highly efficient strategy, based on proteomic analysis and elution chromatography, by which a protein of interest may be isolated from copurifying contaminants. Mutant strains of Escherichia coli were prepared that are deficient in three prevalent host proteins found in a strategic fraction of an elution profile of nickel immobilized affinity chromatography. Recombinant green fluorescent protein (GFPuv) served as a model protein and its elution was directed to this optimized fraction with an N-terminus hexahistidine tag (his6), thereby easing its recovery. We demonstrate that proteomic data can facilitate the rational engineering of host cell expressing the target protein and the design of an efficient process for its purification. |
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Keywords: | Protein purification Immobilized metal affinity chromatography E coli host cell protein contamination Proteomics Host cell design GFP Affinity tag |
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