Proteins have various mechanisms of action, structures and cellular functions. To carry out a function, proteins bind other biomolecules or ligands. Through purifying proteins that are bound to their substrate, researchers can gain valuable insights into how proteins work in a cellular environment. Common techniques for further analyzing protein:ligand complexes to gain structural information include X-ray crystallography, NMR, and cryo-EM. However, a lack of reproducible and robust separation techniques means the purification of protein:ligand complexes remains a challenge.
In this article, a simple, fourth method for gradient preparations is presented. The method uses Beckman Coulter's Biomek 4000 Workstation, which provides reproducible and consistent results in layering discontinuous density gradients, thereby reducing user variability.
Conventional Techniques for Protein Purification
Although linear or continuous rate-zonal density gradients are produced in a number of ways, layering a discontinuous/step gradient is always the first step in the process. The most commonly used technique involves pipetting an aliquot of a less dense solution into a centrifuge tube, followed by the introduction of successively denser solutions to the bottom of the tube using a syringe, so as not to disturb the previous layer and leave a sharp interface between the different density layers.
Another method involves gently layering decreasingly dense solutions on top of a more dense solution. To create a continuous gradient from a discontinuous one, three main methods are used: 1) incubating at 4° to 8°C for 16h or overnight; 2) spinning in a centrifuge for a fixed duration and speed; or 3) using a commercial gradient maker, which spins the tube at a particular angle for a fixed speed and duration. Whichever of these techniques is used, the solution diffuses in such a way that a gradual increase in density is produced from the top of the tube to the bottom.
Both of these layering techniques are time-consuming, tedious to perform and often not reproducible, meaning a lot of practice and patience is required of researchers if they are to produce strong interfaces between the different densities.
Novel Method for Gradient Preparations
The Biomek 4000 Workstation is easy to use, as well as offering excellent precision in liquid handling. In addition, the workstation comes with a cooling, static peltier Automated Labware Position (ALP) that provides an on-deck incubation platform for the linear gradient formation overnight, without a refrigerator or cold room being needed.
Biomek 4000 Workstation
The gradients are fractionated following centrifugation. The fractionation is carried out by either manual pipetting from the meniscus or by making a hole at the bottom of a tube and collecting a specific number of drops per aliquot. Some commercially available systems can perform automated fractionation, but they are usually expensive and also incompatible with some tube types. The Biomek 4000 Workstation ensures accurate liquid handling and was therefore assessed to find out whether it is also suitable for fractionating a sucrose gradient.
ATPase of phi29 and DNA ligand
During maturation, the genomic double stranded (ds) DNA of dsDNA viruses is packaged into a pre-formed protein shell known of as a procapsid. A nanomotor that uses ATP as an energy source completes this entropically unfavorable process. Bacteriophage phi29 is a widely researched phage, owing to its simple structure, which is made up of an ATPase packaging enzyme (gp16), a connector portal protein (gp10) and packaging RNA (pRNA). Guo et al. initially suggested that the mechanism by which dsDNA is packaged into the procapsid is similar to the action of other AAA+ (ATPases Associated with Diverse Cellular Activities) proteins that use ATP as an energy source. In recent years, gp 16 has been found to use a sequential action mechanism with dsDNA and ATP to carry out packaging. In addition, as with other AAA+ proteins, gp16 was also validated as existing as a hexamer on the viral packaging motor and cooperativity was found to exist between the ATPase and ATP, generating a high affinity state for dsDNA following the binding of y-S-ATP, a non-hydrolyzable ATP substrate. Therefore, a revolutionary, new DNA packaging mechanism was proposed and demonstrated. This motor is of particular interest to researchers because it has been widely used in several nanotechnology applications.
For studying this motor, it was necessary to find out how certain components interact with each other in cellular conditions, so as to better understand the phage’s mechanism and biology. In one such assay, the gp16/dsDNA complex was isolated through rate-zonal centrifugation. In a previously published experiment (see Nucleic Acids Research), complexes were purified in a 5-20% sucrose gradient in a Beckman Coulter SW-55 rotor spinning at 35,000rpm. Further kinetic analysis was performed to measure the ATP hydrolysis rate. Additional investigations of the purified complex led to valuable insights that enabled researchers to understand the DNA packaging mechanism in phi29 phage maturation.
In the following example, purification of the gp16/dsDNA complex will be assayed by copying the previously published experiment, but it will be performed using a larger rotor and an automated layering fractionating technique with the Biomek 4000 Workstation.
The method involves diluting sucrose at 5% and 20% (w/v) with a dilution buffer (50 mM NaCl, 25mM Tris pH 8.0, 2% glycerol, 0.01% Tween-20, 2mM MgCl2, 0.15mM y-S-ATP). A gradient was made either manually, by layering 5% solution on top of 20% solution using a pipette or it was made automatically using the Biomek 4000 Workstation. The solutions were incubated overnight at 4°C overnight in a refrigerator or using the pre-cooled peltier ALP of the Biomek 4000 Workstation. For the latter method, 5% or 20% sucrose solutions contained in 15mL conical tubes in a holding rack were placed on-deck, together with a pre-chilled static peltier holding up to six 13.2mL Beckman Coulter polypropylene centrifuge tubes.
The workstation was initially instructed to add 5.5mL of 5% solution to the bottom of the centrifuge tube using a P1000SL tool, in 916.6µl aliquots, followed by the addition of 5.5mL of 20% sucrose beneath the 5% solution. For this step, use of the long and cylindrical Beckman Coulter Span-8 P1000 pre-sterile tips (P/N B01124) minimizes damage to the sucrose interface. The temperature was maintained overnight by the peltier ALP, enabling the sucrose to generate a linear gradient.
In 2009, Gp16 was re-engineered to include a fluorescent arm and referred to as enhanced green fluorescent protein (eGFP). This did not have any effect on the folding or activity of the protein, but it did provide a marker that could be easily identified for use in in vitro and single molecule assays. It has also been demonstrated that eGFP-gp16 binds non-specifically to dsDNA and fluorescent tags such as cy3, can easily be conjugated for further determination. Integrated DNA Technologies (IDT) supplied ultrapurified 40 bp cy3-conjugated dsDNA which was resuspended in DEPC-H2O and Roche Diagnostics supplied y-S-ATP.
The sample preparation was carried out by mixing eGFP-gp16, cy3-dsDNA and, y-S-ATP in final concentrations of 1μm, 250nM and 1.25mM, respectively. The samples were gradually added to the top of the gradient without disturbing the formed gradient. They were then balanced and positioned in a Beckman Coulter Optima XPN Ultracentrifuge where they were spun at 40,000rpm for 7h at 4°C.
The fractionation of samples was then performed, either by fractionating directly from the top of the tube into an opaque microplate using a pipette set at 250µl or by using the Biomek 4000 Workstation to liquid level track the meniscus and automatically transfer the same volume fractions to the microplate. In the Biomek 4000 Workstation technique, a rack carrying the spun centrifuge tubes was placed on-deck, together with a black-bottom microplate. P1000 tips were used to fractionate 250µl from the top of the meniscus and add it to consecutive wells in the 96-well microplate. Based on user-defined parameters for the tube geometry, the Biomek 4000 Workstation can accurately track the liquid level of the tube, while fractions are removed.
A Molecular Devices SpectraMax® i3 Multi-Mode Detection Platform was then used to analyze the fractions at GFP and cy3 wavelengths and the data was exported to a Microsoft® Excel® file for analysis and then transferred into Origin Pro v9.0 for plotting.
Figure 1 shows the plot of data obtained from the Molecular Devices SpectraMax® i3 microplate reader and overlaid for both methods and wavelengths. As fractions were taken from the top of the tube, the sedimentation direction is from left to right on the graphs. The red GFP line represents eGFP-gp16 and the black cy3 line represents cy3-DNA. Figure 1a shows it was possible to resolve free protein and DNA (fraction 2-6) from the protein-DNA complex (fractions 17-32) using the manual layering and manual fractionation technique. Figure 1b shows the free protein and free DNA (fractions 3-6) were separated from the gp16/DNA complex (fractions 17-27) using the Biomek 4000 Workstation for both layering and fractionation. Using the Biomek technique, however, two distinct peaks exist in the complex region, which are particularly evident at the cy3 signal.
It is clear that fractions 16-21 are an independent peak from fractions 24-27, suggesting the existence of two separate conformations or oligomeric state of the protein bound to DNA. This phenomenon has already been mentioned in a recent Virology publication and is a valuable finding, providing important, relevant information about the packaging mechanism. gp16 is thought to first bind to dsDNA as a dimer, followed by assemblage into a hexamer as the last stage of the packaging function. Hypotheses propose that fractions 16-21 represent the dimeric state, while fractions 24-27 consist of the hexamer.
Figure 1a and 1b. Manual versus Biomek 4000 Workstation preparation of a 5–20% linear sucrose gradient.
Figure 2 was plotted using the same data, but the two methods were compared in separate graphs for each signal. The similarity of the overall plot shape suggests a robust automation method that could replace the manual method. Again, it appears that the resolution for the Biomek 4000 Workstation method (red line) is greater, which can be accounted for by improved pipetting methods and decreased physical movement following layering. Also, the standard deviation is comparable between the two techniques, indicating reproducibility.
Figure2a and 2b. Overlaid images of different preparation techniques for eGFP-gp16 (a) and cy3-dsDNA (b).
The purification of protein:ligand complexes is vital for providing insights into biological processes. Purified complexes are often applied in downstream analyses including crystallography, sequencing and high-resolution imaging to investigate protein-based therapeutics. The purified gp16/dsDNA complex in the previous example demonstrated the unique DNA packaging mechanism in a bacteriophage.
The unavailability of a robust, reproducible method for protein:ligand purification has meant research has been limited for many years. Although the use of chromatography has a number of benefits, the technique involves low resolution, protein incompatibilities and large dilution factors. By contrast, density gradient centrifugation, based on the laws of thermodynamics, allows quick modification of parameters, thereby enabling efficient separations.
In this article, an automated rate-zonal centrifugation method is described for purifying protein:ligand complexes. It must be noted that through optimization of spin time, speed and gradient conditions, this method is suitable for almost all proteins. This protocol provides various benefits over the manual method and these are described below.
In scientific experiments that involve layering and fractionating a density gradient by multiple users in a laboratory, human error is common. A steady hand and much patience is required to manually layer a gradient, whether a pipette or the syringe and needle method is used. The Biomek 4000 Workstation offers a distinct interface and consistent fraction at all times, by automating this process. The need to add samples to the same well is eliminated, as is the manual counting of irregular drops from the bottom of a centrifuge tube. Moreover, the automated approach consists of a chilled peltier step that minimizes the jostling of tubes that occurs when gradients are moved to the refrigerator or cold room. This is an important advantage because movement often causes turbidity of the interface.
Ease of Use
Unlike the tedious manual process, the Biomek machine carries out the majority of the work involved in layering and fractionating a density gradient, at the press of a button.
The time taken by the automated method to layer and fractionate a gradient was around the same as the manual approach. The layering of two gradients was achieved in less than 20mins and fractionation of two tubes was achieved in less than 50mins. The difference is that the user can leave the Biomek machine to carry out the main bulk of the work, thereby freeing them up to get on with other tasks.
The Biomek Automated Liquid Handling product line can be used with different types of downstream analysis equipment including microplate readers such as the Molecular Devices SpectraMax® i3 Multi-Mode Detection Platform. This enables researchers to concentrate on more important issues, such as grant writing and data analysis.
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