Use of proteomics for design of a tailored host cell for highly efficient protein purification.

After some initial optimization, a downstream process comprised of one or several chromatography steps removes the majority of the host proteins and achieves a reasonable degree of purification. The separation of remaining contaminant proteins from the target protein could become very difficult and costly due to their similar physicochemical properties. In this paper we describe a highly efficient strategy, based on proteomic analysis and elution chromatography, by which a protein of interest may be isolated from copurifying contaminants. Mutant strains of Escherichia coli were prepared that are deficient in three prevalent host proteins found in a strategic fraction of an elution profile of nickel immobilized affinity chromatography. Recombinant green fluorescent protein (GFPuv) served as a model protein and its elution was directed to this optimized fraction with an N-terminus hexahistidine tag (his(6)), thereby easing its recovery. We demonstrate that proteomic data can facilitate the rational engineering of host cell expressing the target protein and the design of an efficient process for its purification.

[1]  R. Koepsel,et al.  Design of Affinity Tags for One‐Step Protein Purification from Immobilized Zinc Columns , 2000, Biotechnology progress.

[2]  K. Terpe.  Overview of tag protein fusions: from molecular and biochemical fundamentals to commercial systems , 2002, Applied Microbiology and Biotechnology.

[3]  P. Blum,et al.  Physiological consequences of DnaK and DnaJ overproduction in Escherichia coli , 1992, Journal of bacteriology.

[4]  D. Waugh,et al.  Making the most of affinity tags. , 2005, Trends in biotechnology.

[5]  E. Cabiscol,et al.  Novel Antioxidant Role of Alcohol Dehydrogenase E from Escherichia coli* , 2003, Journal of Biological Chemistry.

[6]  Sudhir Sahdev,et al.  Production of active eukaryotic proteins through bacterial expression systems: a review of the existing biotechnology strategies , 2007, Molecular and Cellular Biochemistry.

[7]  Frank Buchholz,et al.  A new logic for DNA engineering using recombination in Escherichia coli , 1998, Nature Genetics.

[8]  F. Baneyx.  Recombinant protein expression in Escherichia coli. , 1999, Current opinion in biotechnology.

[9]  E K M Ueda,et al.  Current and prospective applications of metal ion-protein binding. , 2003, Journal of chromatography. A.

[10]  J. Sambrook,et al.  Molecular Cloning: A Laboratory Manual , 2001 .

[11]  J. V. Van Alstine,et al.  Multipurpose peptide tags for protein isolation. , 2008, Journal of chromatography. A.

[12]  G. G. Stokes.  "J." , 1890, The New Yale Book of Quotations.

[13]  G. Chaga,et al.  Twenty-five years of immobilized metal ion affinity chromatography: past, present and future. , 2001, Journal of biochemical and biophysical methods.

[14]  G. Petersen,et al.  Current strategies for the use of affinity tags and tag removal for the purification of recombinant proteins. , 2006, Protein expression and purification.

[15]  V. Bolanos-Garcia,et al.  Structural analysis and classification of native proteins from E. coli commonly co-purified by immobilised metal affinity chromatography. , 2006, Biochimica et biophysica acta.

[16]  K. Gunsalus,et al.  Protein production and purification , 2008, Nature Methods.

[17]  J. Porath.  Immobilized metal ion affinity chromatography. , 1992, Protein expression and purification.

[18]  W. Hillen,et al.  Quantitative analysis of gene expression with an improved green fluorescent protein. p6. , 2000, European journal of biochemistry.

[19]  R. Beitle,et al.  Genomic data for alternate production strategies. I. Identification of major contaminating species for Cobalt+2 immobilized metal affinity chromatography , 2004, Biotechnology and bioengineering.

[20]  R. Koepsel,et al.  Selection of optimum affinity tags from a phage-displayed peptide library. Application to immobilized copper(II) affinity chromatography. , 1997, Journal of chromatography. A.

[21]  J. Walker,et al.  Over-production of proteins in Escherichia coli: mutant hosts that allow synthesis of some membrane proteins and globular proteins at high levels. , 1996, Journal of molecular biology.

[22]  V. Gaberc-Porekar,et al.  Perspectives of immobilized-metal affinity chromatography. , 2001, Journal of biochemical and biophysical methods.

[23]  Torbjörn Gräslund,et al.  Protein Engineering Strategies for Selective Protein Purification , 2005 .

[24]  R. Gennis,et al.  The sequence of the cyo operon indicates substantial structural similarities between the cytochrome o ubiquinol oxidase of Escherichia coli and the aa3-type family of cytochrome c oxidases. , 1990, The Journal of biological chemistry.

[25]  K. Terpe.  Overview of bacterial expression systems for heterologous protein production: from molecular and biochemical fundamentals to commercial systems , 2006, Applied Microbiology and Biotechnology.

[26]  Caitlin Smith,et al.  Striving for purity: advances in protein purification , 2005, Nature Methods.

[27]  N. Costantino,et al.  Recombineering: in vivo genetic engineering in E. coli, S. enterica, and beyond. , 2007, Methods in enzymology.

[28]  A. Plückthun,et al.  An Escherichia coli protein consisting of a domain homologous to FK506-binding proteins (FKBP) and a new metal binding motif. , 1994, The Journal of biological chemistry.