title: Evaluation of Escherichia coli proteins that burden nonaffinity-based chromatography as a potential strategy for improved purification performance.
abstract: 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.
authors: Department of Bioengineering, 306 Center for Biotechnology, University of Pittsburgh, Pittsburgh, PA 15219, USA.
authors: Patrick
authors: P
authors: Bartlow
authors: Neha
authors: N
authors: Tiwari
authors: Robert R
authors: RR
authors: Beitle
authors: Mohammad M
authors: MM
authors: Ataai
mesh_terms: D002411:Cation Exchange Resins / Q000378:metabolism; D002846:Chromatography, Affinity / Q000379:methods*; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D004926:Escherichia coli / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000302:isolation & purification*; D049452:Green Fluorescent Proteins / Q000302:isolation & purification / Q000378:metabolism; D057927:Hydrophobic and Hydrophilic Interactions; D016297:Mutagenesis, Site-Directed; D010449:Peptide Mapping / Q000379:methods; D040901:Proteomics; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D012269:Ribosomal Proteins / Q000737:chemistry / Q000302:isolation & purification
chemical_list: D002411:Cation Exchange Resins; D029968:Escherichia coli Proteins; D011994:Recombinant Proteins; D012269:Ribosomal Proteins; C031127:ribosomal protein L25; D049452:Green Fluorescent Proteins