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Improving Protein Purification and Biotinylation Workflows

Thu, 08/01/2013 - 1:41pm
Amedeo Cappione, Masaharu Mabuchi, Saosan Suhrawardy, David Briggs, Timothy Nadler, EMD Millipore Corporation

Analyses of protein structure and function depend on the reliable production of pure, functionally active protein, but traditional centrifugation-based affinity purification presents challenges. Batch processing of resins in microcentrifuge tubes is subject to sample loss during aspiration. Spin columns alleviate this concern; however, due to their limited volume capacity, the protocols are tedious, requiring multiple spin cycles. Moreover, while most purification workflows require additional sample neutralization, buffer exchange, and/or concentration prior to downstream analysis, no single traditional centrifugation device can perform all steps.

The Amicon Pro purification system (EMD Millipore) is an adaptable centrifugal device that couples affinity-based spin column purification with downstream sample concentration and buffer exchange. By condensing the protein preparation workflow, the system eliminates the need for multiple sample transfers, minimizing protein loss

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Less time, fewer steps required by the Amicon Pro workflow compared to traditional methods. The table outlines the differences between the two spin-based methodologies at the individual step and overall process levels following purification of a His-tagged protein from 0.5 mL E. coli lysate. The Total time values represent the average and standard deviation of three independent trials involving four devices each.   

The large exchange reservoir accommodates a greater range of sample volumes, permitting rapid purification while reducing centrifugation steps. Direct coupling to Amicon Ultra 0.5 mL centrifugal filters provides simultaneous concentration during the elution phase for compatible sample sizes. The system is also specifically engineered for highly efficient diafiltration in a single spin, further condensing the workflow.

Performance comparison

The Amicon Pro purification system was compared with a traditional 0.5 mL affinity-based spin column. Since these spin columns alone are not able to concentrate or buffer exchange samples, an Amicon Ultra 0.5 mL device was included as part of the spin column workflow.

As shown in Table 1, the new device offered a significant reduction in overall processing time and decreased the number of steps from 31 to 15. One key factor underlying time savings is the reduction in spin steps required during the wash, elution, concentration and buffer exchange phases.

More importantly, the easier process did not compromise sample yield or purity. Given the same starting parameters, the Amicon Pro system yielded 18 percent more His-CRP than the 0.5 mL spin column and 53 percent more His-CRP than the batch slurry method (Figure 1A). One factor contributing to this improved yield was that the Amicon Pro device eliminated sample loss due to transfer between devices. This result also further substantiated the finding that purification using a packed affinity resin is more efficient than in the slurry format where removal of unwanted flowthrough and wash fractions is done through pipeting. The gel image confirmed the purity of the Amicon Pro elution fraction; even with fewer wash steps, no additional contaminating species were found contributing to the final purified sample (Figure 1B).

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Better yield, equal protein purity from Amicon Pro purification compared to two traditional affinity purification schemes. In each case, 100 μL settled resin was used to purify His-CRP from 0.5 mL E coli lysate. (A) The graph shows the difference in total protein yield (His-CRP) from the three different bind-wash-elute methods. Bars represent the average of 12 (Amicon Pro), 6 (0.5 mL spin column), or 4 (slurry method) independent tests. (B) A representative gel showing the various fractions derived during purification using the three methods being compared.  

Efficient washing

Much of the performance improvement can be attributed to the device’s design. The large chamber of the upper exchange device has a 10 mL capacity, which allows wash and elution steps to be achieved with a single one-minute spin. While the system could eliminate nearly all non-target proteins with a single 1.5 mL wash step, the smaller affinity-based spin columns and gravity column-based methods required three washes to clear the resin of nonspecifically bound species (Figure 2). The large chamber is even more advantageous in cases requiring stringent washing, in which the amount of wash buffer used can be increased without increasing process time.

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Efficient clearance of nonspecific protein with a one-spin wash. Ni-NTA agarose resin (100 μL packed volume) was used to purify His-CRP from 0.5 mL lysate using gravity-driven columns or centrifugal devices (n = 3). After unbound protein was cleared (Flowthrough), devices were washed three times (3 mL/wash for the gravity column and Amicon Pro, 0.5 mL/wash for the competitor spin device). Each fraction was collected and total protein content was determined using the Direct Detect™ spectrometer.   

Elution without dilution

Similarly, for the elution phase, a single one-minute spin was sufficient to release >90 percent of bound protein. In fact, the single spin resulted in recovery of more recombinant protein than three elutions from the 0.5 mL spin column. Moreover, when the included Amicon Ultra 0.5 mL filter was attached, the sample underwent simultaneous concentration during the elution step. By combining these two steps, the new device eliminated the requirement of determining protein content across the various eluted fractions before concentrating.

Designed for buffer exchange

Buffer exchange is often required to put protein in the proper context for downstream applications. Dialysis is time-consuming, subject to sample loss, requires large volumes of buffer and necessitates an additional concentration step. Current centrifugal diafiltration methods require multiple rounds of buffering, which increases the potential for protein aggregation and precipitation or loss of structural integrity.

To assess diafiltration performance, equivalent eluted samples of GST-LPP (lambda protein phosphatase; a protein known to lose activity under standard three-spin diafiltration conditions) were buffer-exchanged using the Amicon Pro device (one 15-minute spin using 1.5 mL) or an Amicon Ultra 0.5 mL device (three 15-minute spins using 0.5 mL buffer each). Following exchange, resulting fractions were assayed for phosphatase activity using paranitrophenyl phosphate (pNPP) substrate. Amicon Pro-derived GST-LPP fractions retained higher activity than similar samples buffer-exchanged using a three-spin method. This suggests that the elimination of multiple concentration/dilution cycles had a positive impact on sample integrity.

Antibody biotinylation

Biotin/streptavidin (SA) detection pairs are an effective approach to protein detection that avoids using sec­ondary anti-species antibodies. How­ever, because many biotinylation protocols require buffer exchange prior to and following antibody labeling, the current workflow is time-consuming and subject to protein loss. The Amicon Pro purification system helps address these challenges. Compared to a standard protocol, the system reduced processing time by 70 percent (Table 2).

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Comparison of biotinylation workflows with respect to time and antibody recovery.  

The improved recovery is likely a reflection of sample containment within a single device for the entire process. It may also be caused by the gentler method of buffer exchange. To achieve complete buffer exchange, the smaller diafiltration device used with the traditional biotinylation protocol required multiple concentration/dilution cycles. These multiple cycles may cause either loss of protein function due to destabiliza­tion of the tertiary structure, or physical protein loss due to aggregation and precipitation.

Conclusions

Reliable affinity-based purification is a prerequisite for unlocking the biochemical functionality of proteins. While numerous methods can handle this task, the overall workflow is tedious and suffers from a lack of continuity, increasing the potential for sample loss.

The Amicon Pro device provides a single centrifugal platform for small-scale purification. In comparative analyses, purification of His-tagged protein using the system proved to be faster and easier, and garnered better yields than traditional methods. The device also offers a comparatively streamlined workflow for small-scale antibody labeling, providing significant time-savings when compared to equivalent platforms.

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