Proteome-Based Development of Affinity Motifs and Reduced Contaminant Strains

Thursday, October 20, 2011: 3:15 PM
M100 I (Minneapolis Convention Center)
Ellen M. Brune1, Tammy M. Rechtin1, Alicia Kight2, Mohammad M. Ataai3, Ralph Henry2 and Robert R. Beitle1, (1)Ralph E Martin Department of Chemical Engineering, University of Arkansas, Fayetteville, AR, (2)Department of Biological Sciences, University of Arkansas, Fayetteville, AR, (3)Chemical and Petroleum Engineering, University of Pittsburgh, Pittsburgh, PA

This effort comprises both the development of novel affinity purification tags concurrent with the construction of host cell(s) with an optimized proteome.  Protein affinity tags are designed regions (both short peptides and large independent domains) that enhance a chromatographic process by utilizing the region’s natural propensity to bind to a specific target immobilized on a solid matrix. Affinity tags designed by our group have the ability to promote binding to multiple stationary phases, using the adsorption and elution characteristics of naturally occurring genomic proteins as guideposts.  One such affinity motif has been based on Escherichia coli peptidyl prolyl cis/trans-isomerase (slyD).  The slyD gene product is a known Immobilized Metal Affinity Chromatography (IMAC) and Ion-Exchange Chromatography (IEC) contaminant that significantly reduces column capacity and complicates gradient design.  This genomic protein provided a candidate amino acid sequence, for promoting adsorption during IMAC and IEC, consisting of a 55 amino acid sequence rich in histidine, glutamic acid and aspartic acid.  Two affinity tags were developed and were fused to Green Fluorescent Protein (GFP) along with other proteins to analyze expression, solubility of the fusion product and isolation.  This presentation will discuss the process leading to the design of the affinity sequences, present the cloning strategy for the GFP construct and the optimized cell line, and compare binding characteristics and solubility in the presence and absence of the affinity tags.  Data will be presented and compared other affinity tags (e.g. His6, Arg6 and GST).

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See more of this Session: Bioseparations and Downstream Processing
See more of this Group/Topical: Food, Pharmaceutical & Bioengineering Division