(515s) The Metalloproteome of Escherichia Coli: Its Application in Downstream Processing Via Imac

Escherichia coli expression systems are important tools for structural and functional analysis in fundamental research and as well as production of foreign proteins as inclusion bodies in industry for high throughput (1). Adding an affinity tag is attractive for a target protein with unknown purification characteristics and economically favorable. Indeed, more than 60% of proteins for structural studies are produced with a poly-histidine tag (his-tag). (2). Naturally occurring histidine and cysteine rich regions in host cell proteins result in unwanted protein binding during an Immobilized Metal Affininty Chromatography (IMAC) capture step. (3). Finding information about these host proteins which have affinity to IMAC columns will be helpful to understand and control contaminants that elute with his-tagged proteins.

We identified several proteins of E. coli that bind to IMAC columns under different conditions. This data helps to choose best combination of support, metal and binding conditions for a protein to be expressed. Selection of intermediate and polishing purification steps can be done based on the properties of contaminants. A three fold strategy has been developed to improve purification processes: mutation of genes corresponding to essential proteins, deletion of genes corresponding to non essential proteins, and rational affinity tail design to move the target elution into a region of less contaminant. During this presentation we will discuss the use of this information to design E. coli strains and use them during the expression of a recombinant protein.

(1)Schmidt F.R. ?Recombinant expression systems in the pharmaceutical industry?, Appl Microbiol Biotechnol 65: 363-372, 2004. (2)Z.S. Derewenda, ?The use of recombinant methods and molecular engineering in protein crystallization?, Methods 34 (2004) 354?363. (3)D.F. Westra, et al., ?Immobilised metal-ion affinity chromatography purification of histidine-tagged recombinant proteins: a wash step with a low concentration of EDTA?, J. Chromatogr. B Biomed. Sci. Appl. 760 (2001) 129?136.