Isolation of recombinant proteins from culture broth by co‐precipitation with an amino acid carrier to form stable dry powders |
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Authors: | Barry D Moore Joseph Deere RuAngelie Edrada‐Ebel Andrew Ingram Christopher F van der Walle |
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Institution: | 1. Department of Pure and Applied Chemistry, University of Strathclyde, Thomas Graham Building, 295 Cathedral Street, Glasgow G1 1XL, UK, telephone: +44‐141‐548‐2301;2. fax: +44‐141‐548‐4822;3. Strathclyde Institute of Pharmacy and Biomedical Sciences, University of Strathclyde, 27 Taylor Street, Glasgow G4 0NR, UK, telephone: +44‐141‐548‐5755;4. fax: +44‐141‐552‐2562 |
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Abstract: | Protein‐coated microcrystals can be generated by co‐precipitation of protein and a water‐soluble crystalline carrier by addition to excess water miscible organic solvent. We have investigated this novel process for its utility in the concentration and partial purification of a recombinant protein exported into the culture broth during expression by Pichia pastoris. Co‐precipitation with a L ‐glutamine carrier selectively isolated the protein content of the culture broth, with a minimal number of steps, and simultaneously removed contaminants including a novel yeast metabolite. This pigment co‐elutes during aqueous chromatography but its elucidation as a benzoylated glycosamine suggested a simple route of removal by partition during the co‐precipitation process. Scale‐up of the process was readily achieved through in‐line mixing and subsequent reconstitution of the dried protein‐coated microcrystals yielded natively folded, bioactive protein. Additional washing of the crystals with saturated L ‐glutamine facilitated further purification of the recombinant protein immobilized on the L ‐glutamine carrier. Thus, we present a novel method for the harvesting of recombinant protein from culture broth as a dry powder, which may be of general applicability to bioprocessing. Biotechnol. Bioeng. 2010;106: 764–773. © 2010 Wiley Periodicals, Inc. |
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Keywords: | protein coated micro‐crystals immobilization concentration purification bioprocessing yeast metabolite |
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