title: A redesigned Escherichia coli triosephosphate isomerase restores growth properties in a bacterial strain useful for Immobilized Metal Affinity Chromatography (IMAC).
abstract: The bacterium Escherichia coli is one of the most commonly used organisms in biotechnology for recombinant protein production and high-throughput development of biopharmaceuticals. The focus of this article is the utilization of proteome based data to design an E. coli expression strain that is improved for initial protein capture via Immobilized Metal Affinity Chromatography (IMAC). Proteome data was specifically applied to guide the modification of a known IMAC binding protein, triosephosphate isomerase (tpiA gene product), and the use of site directed mutagenesis eliminated binding properties. The designer tpiA gene, when reintroduced into an E. coli strain deficient in this enzyme activity, produced a functional protein lacking in surface exposed histidine and was able to restore glycolytic function.
authors: Ralph E. Martin Department of Chemical Engineering, 3202 Bell Engineering Center, University of Arkansas, Fayetteville, AR 72701, United States.
authors: R
authors: R
authors: Haley
authors: Boston Mountain Biotech, LLC 534W Research Center Blvd, Suite 254, Fayetteville, AR 72701, United States.
authors: M
authors: M
authors: Fruchtl
authors: Boston Mountain Biotech, LLC 534W Research Center Blvd, Suite 254, Fayetteville, AR 72701, United States.
authors: E M
authors: EM
authors: Brune
authors: Department of Chemical and Petroleum Engineering, University of Pittsburgh, 1249 Benedum Hall, Pittsburgh, PA 15261, United States.
authors: M
authors: M
authors: Ataai
authors: Department of Biological Sciences, 601 Science Engineering, University of Arkansas, Fayetteville, AR 72701, United States.
authors: R
authors: R
authors: Henry
authors: Ralph E. Martin Department of Chemical Engineering, 3202 Bell Engineering Center, University of Arkansas, Fayetteville, AR 72701, United States. Electronic address: rbeitle@uark.edu.
authors: R
authors: R
authors: Beitle
mesh_terms: D000595:Amino Acid Sequence; D002846:Chromatography, Affinity / Q000379:methods*; D004926:Escherichia coli / Q000201:enzymology* / Q000254:growth & development; D008670:Metals / Q000737:chemistry; D008969:Molecular Sequence Data; D014305:Triose-Phosphate Isomerase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: D008670:Metals; D014305:Triose-Phosphate Isomerase
keywords: Escherichia coli; Immobilized metal affinity chromatography; PCR mutagenesis; Protein engineering; Triosephosphate isomerase