December 1, 2014 (Vol. 34, No. 21)

Anja Dedeo senior scientist EMD Millipore
Jesmi George research scientist EMD Millipore

New One-Step Protocol Saves Time and Delivers Greater Consistency

Traditionally, 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).

Incorporation of magnetic beads into the protocol eliminates the need to clarify lysates by centrifugation. Magnetic beads have been adopted where agarose beads have been used, reducing processing time and increasing sample throughput. Magnetic beads are generally used in batch mode and isolated on a magnet to allow for buffer exchange.

Magnetic beads facilitate automation of the protocol using a particle processor, resulting in increased sample throughput while reducing hands-on processing time to less than 10 minutes. To further improve the workflow, we used BugBuster® Master Mix reagent (EMD Millipore), which allows for nonmechanical extraction of soluble protein from bacterial cells. This extraction reagent combines detergent-based lysis with the enzymatic agent Benzonase® nuclease and the enzyme rLysozyme™ in a ready-to-use formulation.


Figure 1. One-step lysis combined with purification (right) saves considerable time compared to traditional recombinant protein purification, which requires separate lysis, lysate clarification, and purification steps (left).

Materials and Methods

Histidine-tagged recombinant glyceraldehyde phosphate dehydrogenase (GAPDH) was purified with PureProteome™ Nickel Magnetic Beads (EMD Millipore), using a traditional purification workflow (mechanical lysis) and the new condensed protocol (combined detergent-based lysis and purification). These magnetic beads capture histidine-tagged proteins; they isolate recombinant proteins at high purity and can be used manually or on automated systems. We compared the manual processing results with those obtained using the KingFisher® automated particle processor (Thermo Scientific).

Traditional Protein Purification
E. coli culture was pelleted into microcentrifuge tubes and the supernatant was discarded. Lysis/wash buffer containing lysozyme was added to each pellet. The pellet was resuspended and incubated with end-over-end mixing, followed by sonication using a microtip. The lysate was frozen, followed by quickly thawing; the sonication/freeze-thaw cycle was repeated once more. To reduce viscosity, Benzonase endonuclease was added to the lysate and clarified by centrifugation.

The clarified lysate was added to PureProteome Nickel Magnetic Beads. The beads were incubated with E. coli lysate with end-over-end mixing. After removal of the lysate, the beads were washed with lysis/wash buffer by vortexing, capturing the beads on the magnet and removing the buffer by pipette. The wash step was repeated two more times prior to eluting the captured histidine-tagged GAPDH. The beads were mixed, and an additional elution was performed to achieve maximum yield. Both elution fractions were combined into one microcentrifuge tube for future analysis.

One-Step Protein Purification
Manual Processing
E. coli culture was pelleted into microcentrifuge tubes and the supernatant discarded. Suspended PureProteome Nickel Magnetic Bead slurry was added. Using the PureProteome Magnetic Stand, the preservative was removed, and the beads were washed with lysis/wash buffer. The beads were collected on the magnet, and the buffer was removed by pipette. The beads were resuspended in BugBuster Master Mix and the suspension was added to each E. coli pellet. Each tube was briefly vortexed, then incubated with end-over-end mixing. The unbound fractions were discarded and beads were washed using the PureProteome Magnetic Stand to capture beads. Elution was performed as previously described.

Automated Processing
E. coli culture was pelleted into a KingFisher Microtiter Deepwell 96 plate and the supernatant discarded. Reagents and samples were pipetted into the KingFisher Duo plates and the suspended PureProteome Nickel Magnetic Bead slurry was brought up to sufficient volume with wash buffer. Wash buffer was used for both equilibration and wash steps, and the elution buffer was pipetted into the elution strips. The protocol was executed and the plates were loaded into the KingFisher Duo System.

The PureProteome Nickel beads were equilibrated in wash buffer to remove preservatives. The beads were collected, and cell lysis and His-tagged protein capture was performed. The beads were then washed and recombinant protein was eluted. After the run ended, the eluted sample fractions were collected for further analysis.

Protein Analysis
Additional lysates were prepared using the traditional mechanical lysis approach as well as using the BugBuster Master Mix protocol; no magnetic beads were added during the lysis step. The lysate was clarified by centrifugation, and total protein concentration was determined with the Direct Detect®
IR spectrometer (EMD Millipore).

A quantitative Bradford assay was also performed to assess the efficiency of lysis and protein purification.

Finally, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was performed. Purified samples were reduced, denatured and loaded onto NuPAGE® Bis-Tris gels (Life Technologies). Following electrophoresis, the gels were stained with Coomassie® blue to visualize protein bands.

Results

The results demonstrated that the new protocol delivered more total protein in less time and with greater consistency than the traditional method. Traditional mechanical lysis of six samples took about two hours; the average total protein collected was 3.54 mg/mL and the CV% was 15.55. In contrast, enzymatic lysis using the new protocol took about 30 minutes; the average total protein collected was 4.09 mg/mL and the CV% was 2.05.

The eluted fractions were then tested for protein yield using a Bradford assay. All workflows demonstrated roughly equivalent yields of purified protein. However, the traditional method exhibited greater inter-sample variability than did either condensed protocol. Workflow automation offered the highest degree of reproducibility (Table).

Finally, the SDS-PAGE gel showed all workflows provided similar sample purity (Figure 2). As previously noted, greater sample-to-sample variability was observed for the traditional workflow.


Yield of His-tagged GAPDH using the traditional and condensed E. coli lysis and purification protocol as determined by Bradford assay.

Discussion

For extraction of recombinant protein from E. coli, traditional mechanical lysis is a time-consuming manual process. Mechanical lysis and manual processing can also result in variable yield due to sample-to-sample variations in the sonication step. By combining gentle nonmechanical lysis with magnetic affinity capture beads for His-tagged protein purification, the traditional recombinant protein purification workflow has been condensed.

Even when samples were manually processed, a one-step lysis and purification protocol reduced processing time by 75% without sacrificing yield or purity. Due to reduced sample manipulation, the simplified protocol also provides greater sample-to-sample consistency. Moreover, by eliminating centrifugation requirements, the condensed workflow can be automated using particle processors to further reduce sample variability and increase throughput while reducing hands-on time to less than 10 minutes.


Figure 2. SDS-PAGE analysis of His-tagged GAPDH purified using the traditional and condensed recombinant protein purification workflows. The condensed protocol was performed manually as well as on the KingFisher Duo System. (Molecular weight standards are included in the rightmost lane.)

Anja Dedeo (anja.dedeo@emdmillipore.com) is senior scientist and Jesmi George (jesmi.george@emdmillipore.com) is research scientist at EMD Millipore.