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Evaluation of Escherichia coli proteins that burden nonaffinity‐based chromatography as a potential strategy for improved purification performance
Author(s) -
Bartlow Patrick,
Tiwari Neha,
Beitle Robert R.,
Ataai Mohammad M.
Publication year - 2011
Publication title -
biotechnology progress
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.572
H-Index - 129
eISSN - 1520-6033
pISSN - 8756-7938
DOI - 10.1002/btpr.703
Subject(s) - escherichia coli , ion chromatography , mutagenesis , affinity chromatography , downstream processing , green fluorescent protein , chemistry , recombinant dna , chromatography , protein purification , host (biology) , biochemistry , biology , computational biology , enzyme , gene , genetics , mutation
Escherichia coli is a favored host for rapid, scalable expression of recombinant proteins for academic, commercial, or therapeutic use. To maximize its economic advantages, however, it must be coupled with robust downstream processes. Affinity chromatography methods are unrivaled in their selectivity, easily resolving target proteins from crude lysates, but they come with a significant cost. Reported in this study are preliminary efforts to integrate downstream separation with upstream host design by evaluating co‐eluting host proteins that most severely burden two different nonaffinity‐based column processes. Phosphoenolpyruvate carboxykinase and peptidase D were significant contaminants during serial purification of green fluorescent protein (GFP) by hydrophobic interaction and anion exchange chromatography. Ribosomal protein L25 dominated non‐target binding of polyarginine‐tagged GFP on cation exchange resin. Implications for genetic knockout or site‐directed mutagenesis resulting in diminished column retention are discussed for these and other identified contaminants. © 2011 American Institute of Chemical Engineers Biotechnol. Prog., 2012