BIOT 77 |
| Immobilized metal affinity chromatography (IMAC) is the most widely used technique for in purification of recombinant proteins. Although its use in bioseparation is pervasive, the true bottleneck with IMAC now lay with the presence of native E. coli proteins that show a high affinity for divalent cations like nickel, cobalt or zinc. Naturally occurring histidine and cysteine rich regions in the proteins of the host cell result in unwanted protein binding during the capture step. Profiling these host proteins which have affinity to IMAC columns will be helpful to understand and control contaminants that elute with target proteins. We identified several proteins of E. coli bind to IMAC columns under different conditions. This data helps to choose best combination of support, metal and binding conditions for a target protein to be expressed. Selection of intermediate and polishing purification steps can be done based on the properties of contaminants to achieve high purities (e.g. 99.5 or 99.8%). A three fold strategy has been developed to improve purification processes: (1) mutation of genes corresponding to essential proteins, (2) deletion of genes corresponding to non essential proteins, and (3) 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. |
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Downstream Processing: Advances in Chromatography
8:00 AM-11:00 AM, Monday, August 20, 2007 BCEC -- 107 A/B, Oral
Division of Biochemical Technology |