Many scientists require purified proteins for their research applications. To meet this need, a multitude of recombinant expression techniques have been developed. While many expression systems are available, E. coli represents the most optimized and widely used vehicles for the expression of 6x Histidine tagged recombinant proteins. Traditionally, the recombinant protein purification workflow consists of five distinct phases: Harvest, Lysis, Clarification, Purification and Analysis. Lysis typically requires multiple time consuming, hands‐on steps, such as freeze/thaw cycling and sonication, which may be harsh and may negatively impact protein quality and contribute to sample variability. Clarification is typically performed in a centrifuge, which makes automation challenging and is primarily needed to reduce clogging in the downstream purification step. Purification is typically performed using columns packed with agarose beads with specificity for the tag on the recombinant protein. While columns provide an easy approach for manipulation of the affinity media, they are greatly affected by lysate consistency and carry‐over of cell debris, which can lead to clogging and poor protein purification and recovery. To guarantee maximum recovery of protein activity and integrity, we have developed a protocol leveraging gentle enzymatic cell lysis and purification with magnetic beads to condense the traditional His‐tag protein purification workflow. A significantly improved workflow was developed by combining Lysis and Purification and eliminating the need for Clarification, resulting in a greater than two hour time savings over the traditional workflow. In addition, this condensed workflow facilitates automation, resulting in increased sample throughput while further reducing actual hands‐on processing time.
raditionally, protein purification from E. coli includes four phases: harvest, bacterial cell lysis, lysate clarification, and protein purification. Bacterial lysis typically requires several time-consuming steps, including freeze/thaw cycles and sonication, which may negatively impact protein quality and contribute to sample variability. To maintain protein activity and integrity, detergent-based lysis buffers are used to avoid mechanical protein extraction methods. Centrifugation is traditionally required to pellet unwanted cell debris and permit clarified lysate recovery.Purification is frequently performed using affinity media specific for expressed epitope tags. Agarose-based media are typically used, either as a slurry in microcentrifuge tubes or packed into columns. While easier to manipulate, columns are affected by lysate consistency and carryover of cell debris, which can cause clogging. This article will demonstrate a new protocol for streamlining the traditional recombinant protein purification workflow by combining the enzymatic lysis and purification steps. This approach results in significantly less hands-on time and greater than two-hour time savings over the traditional workflow (Figure 1). Anja Dedeo (anja.dedeo@emdmillipore.com) is senior scientist and Jesmi George (jesmi.george@emdmillipore.com) is research scientist at EMD Millipore. Website: www.emdmillipore.com. T
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