Use of proteomics for design of a tailored host cell for highly efficient protein purification.
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 (his(6)), 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.
Liu
Zhu
Z
Department of Chemical and Petroleum Engineering, University of Pittsburgh, Pittsburgh, PA 15261, USA.
Bartlow
Patrick
P
Varakala
Rajaramesh
R
Beitle
Robert
R
Koepsel
Richard
R
Ataai
Mohammad M
MM
D002846:Chromatography, Affinity / Q000379:methods*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000235:genetics*; D055786:Gene Knockout Techniques / Q000379:methods*; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D006639:Histidine / Q000235:genetics / Q000378:metabolism; D009154:Mutation; D009532:Nickel / Q000737:chemistry; D009842:Oligopeptides / Q000235:genetics / Q000378:metabolism; D011506:Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D040901:Proteomics / Q000379:methods*; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism
D029968:Escherichia coli Proteins; C471213:His-His-His-His-His-His; D009842:Oligopeptides; D011506:Proteins; D011994:Recombinant Proteins; D049452:Green Fluorescent Proteins; D006639:Histidine; D009532:Nickel