Evaluation of Escherichia Coli Proteins That Burden Recombinant Protein Purification On Inexpensive, Non-Affinity-Based Resins

Wednesday, October 19, 2011: 3:15 PM
Conrad A (Hilton Minneapolis)
Patrick R. Bartlow1, Neha Tiwari2, Robert R. Beitle2 and Mohammad M. Ataai3, (1)Bioengineering, University of Pittsburgh, Pittsburgh, PA, (2)Ralph E. Martin Department of Chemical Engineering, University of Arkansas, Fayetteville, AR, (3)Chemical and Petroleum Engineering, University of Pittsburgh, Pittsburgh, PA

Escherichia coli has long been a favored host for rapid, scalable expression of recombinant proteins for commercial or academic use. Upstream advantages must be coupled with robust downstream processes, however, to maximize its economic benefits. While affinity chromatography is unrivaled in its selectivity and resolving capabilities, it comes with significant cost. This presentation reports on preliminary efforts to integrate downstream processing with upstream host design by evaluating co-eluting host proteins that most severely burden purification by non-affinity-based column processes. Phosphoenolpyruvate carboxykinase and peptidase D were significant contaminants during serial purification of GFPuv by hydrophobic interaction and anion exchange chromatography. With the intent to improve downstream efficiency with cheaper resins via host modifications, implications for genetic knockout or site-directed mutagenesis that diminishes contaminant retention are discussed for these and other identified host proteins.

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