(556a) Knockout and Mutational Tailoring of Host Strains to Simplify Bioseparation

This presentation will outline our efforts to deploy the combination of proteomics and molecular biology for the improvement of bioseparation and illustrate this useful pairing for strain development to minimize difficulties associated with copurifying contaminants. Separation of a target recombinant protein and host cell protein(s) becomes difficult when similar physicochemical properties are displayed. This difficulty manifests itself in the loss of column capacity when the target protein is expressed at levels comparable to the genomic contaminants, and in the tailoring of gradient when differential elution becomes necessary. A novel strategy would be to employ proteomics to identify contaminants that may be strategically removed from the genome or altered in order to simplify bioseparation. Several mutations in Escherichia coli have been designed and tested with the intention of illustrating this strategy and improving Immobilized Metal Affinity Chromatography (IMAC). Specifically, (i) a functional version of the enzyme triosephosphate isomerase has been redesigned with three point substitutions that abolish IMAC retention and maintain glycolysis, and (ii) three proteins (gene products of cyoA, yfbG, and adhP) have been deleted from the window within an imidazole gradient that contains the fewest copurifying contaminants. Combined, these changes result in an IMAC-optimized E. coli that facilitated the recovery of Green Fluorescent Protein as an illustrative recombinant product.