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A redesigned Escherichia coli triosephosphate isomerase restores growth properties in a bacterial strain useful for Immobilized Metal Affinity Chromatography (IMAC)

Haley, R., Fruchtl, M., Brune, E.M., Ataai, M., Henry, R., Beitle, R.
Journal of biotechnology 2014 v.188 pp. 48-52
Escherichia coli, affinity chromatography, bacteria, binding properties, binding proteins, biopharmaceuticals, biotechnology, enzyme activity, genes, glycolysis, histidine, proteome, recombinant proteins, site-directed mutagenesis, triose-phosphate isomerase
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.