7 %B Scouting Identifies Optimal Elution Buffer Concentration and Gradient
Once an optimal pH was established for prancer purple binding, we optimised the elution step to separate prancer purple from remaining contaminants. Using the %B scouting option of ChromLab software, we assayed a 10 CV elution gradient from 20–50% final %B in four separate runs. The shallowest buffer gradient, 0–20% B, resulted in optimal enrichment of prancer purple (Figure 4A, B). Some high and low molecular weight contaminants were still present, leading us to add additional purification steps to our workflow (Figure 4B).
Elimination of Remaining Contaminants Using Bio-Scale Mini CHT Type II Ceramic Hydroxyapatite Resin
During our initial column scouting phase, we also assessed prancer purple binding to the mixed-mode ion exchange resin Bio-Scale Mini CHT Type II ceramic hydroxyapatite. Prancer purple showed high affinity for this resin and markedly, the contaminants in the elution profile were distinct from those found in our Foresight Nuvia Q elution. We therefore chose Bio-Scale Mini CHT Type II resin for our second purification step, to remove remaining contaminants from the Foresight Nuvia Q elution. The optimised Foresight Nuvia Q–Bio-Scale Mini CHT Type II column combination yielded highly pure prancer purple as illustrated by the absence of contaminating bands in Any kD Criterion TGX Stain-Free polyacrylamide gels (Figure 5).
Buffer Exchange and Sample Polishing Using Size Exclusion Chromatography
For some applications, such as crystallography, final elution buffer composition is crucial. Similarly, many applications require the final sample buffer composition to be identical from batch to batch. When using gradient elution, however, the exact buffer composition of the pooled elution fractions is frequently unknown. Size exclusion chromatography (SEC) is an excellent
method to ensure consistent buffer composition from prep to prep, since fractionation is not dependent on buffer gradients. SEC also eliminates low abundance contaminant, proteolyzed fragments, and aggregate species, ensuring high purity and uniformity of final protein. We therefore incorporated a final size exclusion step in our purification protocol using the ENrich SEC 70 column, which has a high pressure limit that allowed higher flow rates and reduced the time needed for this step compared to carbohydrate based beads (Figure 5).
Figure 5. Final purification workflow for untagged prancer purple Any kD Criterion TGX Stain-Free SDS-PAGE gel. 20 µl of sample was loaded. Lysate (L) and Nuvia Q flowthrough were diluted 1:20. Elutions (E) are shown for Nuvia Q, CHT Type II ceramic hydroxyapatite, and ENrich 70 columns. Prancer purple molecular weight: 26.4 kD.
Discussion
This study illustrates that untagged protein can be purified to high homogeneity when purification options are sufficiently explored. Exhaustive exploration is often impractical, because identifying an ideal purification workflow requires optimisation of multiple variables, such as column resin, pH, and %B, at each step. This means that a multitude of runs is required at each purification step to identify the purification workflow that yields protein of the desired purity. Traditionally, this process requires individually setup and programmed runs for each tested column, pH, and %B condition. ChromLab software, however, allowed us to automate and accelerate this tedious process.
ChromLab software can be programmed to execute several runs sequentially. It controls different components of the NGC system, such as the NGC sample pump that directly injects sample onto the column, the buffer blending valve that allows scouting of pH and %B, and the column switching valve that can automatically switch between up to five different columns. This permitted us to scout four ion exchange columns without user intervention in the same setup and instrument programming time as that required for one traditional run. In a similar fashion, we
were able to explore multiple pH conditions without the need to make and titrate individual buffers for each tested pH. Finally, automated %B scouting allowed us to improve the final purity of protein.
Our data show that column scouting is essential, even when a specific resin type, such as anion exchange resin, has already been chosen. As we show here, resins designed for similar use (in this case anion exchange) can show drastically different binding efficiencies for a given protein. Even though Foresight Nuvia Q and Bio-Sclae Mini UNOsphere Q resins are both strong anion exchange resins with trimethylamine functional groups, prancer purple bound only to Foresight Nuvia Q resin. This is most likely due to differences in bead chemistries and size.
A second challenge of purification workflow development is evaluating which of the scouted methods yields the best results. Traditionally, based on the A280 trace of the chromatogram, which tracks the presence of protein eluting from the column throughout the chromatography run, fractions are chosen for analysis by SDS-PAGE. A method is then chosen that (1) yields high amounts of the protein of interest, (2) yields few contaminating proteins, and (3) elutes the protein of interest over a small volume. Here we introduced a third factor, the purity quotient difference (PQD), that can inform method development when the protein of interest and contaminants absorb light at different wavelengths.
Most proteins contain tryptophans and thus absorb light at a wavelength of 280 nm. Chromogenic proteins, fluorescently tagged proteins, or metal-bound proteins such as hemoproteins, as well as contaminants such as DNA, absorb light at other wavelengths. Using the NGC Discover multiwavelength detection system, we could distinguish the elution profile of prancer purple (525 nm) from the elution profile of contaminating proteins (280 nm). Because the area under the absorbance peaks in a chromatogram is proportional to the amount of protein eluted in that peak, we were able to calculate the purity quotient difference for prancer purple.
The peak integration feature of ChromLab software calculates the area under elution peaks, as well as the relative peak area, which is a measure of what percentage of total protein of interest loaded elutes in a particular peak (Figure 2C). Since we wanted to determine which method yielded the highest concentration of prancer purple, we normalised the relative peak area by dividing it by the volume collected. We call this value the purity quotient and it was calculated for prancer purple (PQ525
) and total protein (PQ280 ).
The goal of preparative chromatography, however, is not simply to obtain a high yield of protein; the goal is to obtain a high yield of pure protein. Purity is traditionally assessed qualitatively by SDS-PAGE. By subtracting the purity quotient of the contaminant (PQ280 (PQ525
) from the purity quotient of the protein of interest ), we calculated the purity quotient difference for prancer purple (PQDPP ).
Using these calculations, a PQD of zero indicates that contaminants and the protein of interest are present at equal amounts. A PQD >0 indicates that the protein of interest is enriched, and a PQD <0 indicates that contaminants are enriched. PQDs can be plotted across fractions to generate PQD histograms. PQD histograms for different scouting conditions can then be overlaid to identify the method with the highest PQD over the smallest number of fractions (Figure 2D, 3C, 4C).
PQD analysis, however, is intended to complement, not replace, SDS-PAGE. Although a high PQD indicates enrichment of the protein of interest, it does not preclude the presence of contaminating proteins. The PQD histograms for pH scouting would suggest that there is no improvement in purification when the pH is raised to 8.0 from 7.5 (Figure 3C). SDS-PAGE analysis, however, clearly shows that more contaminants co-elute with prancer purple at pH 8.0 (Figure 3B).
PQD histograms also do not address the nature of contaminants. Our initial round of column scouting included the mixed-mode Bio-Scale Mini CHT Type II ceramic hydroxyapatite resin. We found that prancer purple was purified to a similar extent using the Foresight Nuvia Q and Bio-Scale Mini CHT Type II resins but different contaminating proteins were found in the elutions. We were thus able to identify an optimal second column for prancer purple purification in our initial column scouting runs.
This emphasises the need to analyse not only the final purity of the protein of interest but also the impurities present in each column scouting run, as this information can inform the choice of resin for subsequent purification steps. Using Bio-Rad’s precast Any kD Criterion TGX Stain-Free polyacrylamide gels we were able to resolve and analyse protein samples in less than 30 minutes, which permitted us to characterise elution patterns from a large number of fractions in a very short time.
The combination of SDS-PAGE and PQD analysis described here can be applied to column, pH, and %B scouting, as well as additional scouting steps, such as optimisation of organic phase for reverse-phase chromatography or ammonium sulphate concentration for hydrophobic interaction chromatography. As this prancer purple case study illustrates, NGC system-mediated automation of these scouting processes allowed exhaustive exploration of purification options. Combined with ChromLab software’s intuitive programming features and preloaded purification templates, the NGC system allowed us to purify an untagged protein without compromising purity.
B-PER is a trademark of Thermo Fisher Scientific.
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