AbstractHigh purity protein is a common requirement for biochemical and structural studies. A common approach is to recombinantly express an affinity-tagged version of the protein of interest. This is, however, not always a viable option. In this report we discuss protein purification workflow development for untagged proteins and introduce a new indicator of method performance, the purity quotient difference (PQD). We show a case study on the process of optimization of the purification workflow for the untagged prancer purple protein by scouting for the optimal resin, pH, and %B using the NGC chromatography system.
Column Scouting Fig. 2. Optimization of resin, SDS-PAGE analysis, and evaluation. Final Purification Scheme for Untagged Prancer Purple Protein Fig. 5. SDS-PAGE analysis of the purified untagged prancer purple protein.ChromLab™ software scouting feature, allows us to automate the process of column, pH, and %B optimization. Using the untagged, chromogenic protein, prancer purple, we illustrate that small changes in column chemistry or pH have drastic effects on protein binding and changes in the elution buffer pH or gradient can greatly impact purity of the eluted protein. Our findings cement the importance of column, pH, and buffer scouting when developing purification workflows and illustrate how the Bio-Rad NGC chromatography system and ChromLab software facilitate this process and make untagged proteins of high purity attainable for every researcher. NGC Discover™ system was used for all chromatography. Three 1 ml anion exchange chromatography columns, Foresight™ Nuvia™ Q, Bio-Scale™ Mini UNOsphere™ Q, and Bio-Scale™ Mini Macro-Prep® High Q, and a 5 ml Bio-Scale™ Mini CHT™ Type II chromatography column were attached to different positions on the NGC column switching valve. The ion exchange scouting method was generated in ChromLab software. Using the buffer blending valve, stock solutions Q1 (0.2 M HCl), Q2 (0.2 M Tris), Q3 (water), and Q4 (4 M NaCl) were mixed to generate buffer A (50 mM Tris pH 7.5) and buffer B (50 mM Tris pH 7.5 and 1 M NaCl). The scout feature was used to scout four columns attached to different positions of the column switching valve. Clarified lysate was diluted threefold with water and 1 ml of diluted lysate was directly injected onto the columns using the sample pump. Columns were equilibrated for 5 column volumes (CV) with buffer A. Approximately 1 ml of sample was applied to the columns. The columns were then washed with 5 ml of buffer A and protein was eluted with a 20 CV linear gradient from 0–50% B followed by a 10 CV 50% B wash. After the linear gradient the columns were stripped and re-equilibrated with a 5 CV 100% B wash, followed by a 10 CV buffer A wash. All purification steps were carried out at a 1 ml/min flow rate. Fractions from each run were analyzed by SDS-PAGE. pH Scouting Once the Foresight Nuvia Q column was selected as the first column, the ChromLab software scout feature was set to the pH scouting option. Binding was assessed at pH 7.0, 7.5, 8.0, and 8.5. 1 ml of diluted lysate was directly injected onto the Foresight Nuvia Q column at each pH during the method using the sample pump. Approximately 0.2 ml fractions were collected from each run and analyzed by SDS-PAGE. %B Scouting After pH 7.5 was selected, the scout feature was used to optimize %B. The 10 CV linear gradient portion of the elution phase was selected for the %B scouting (20, 30, 40, and 50 final %B). 1 ml of diluted lysate was directly injected onto the Foresight Nuvia Q column at each pH using the sample pump. 0.2 ml fractions were collected from each run and analyzed by SDS-PAGE. CHT Ceramic Hydroxyapatite Chromatography The pooled Foresight Nuvia Q fractions were diluted twofold with water. The buffer system on the Bio-Rad NGC Discover system was changed to phosphate (Q1 = 0.2 M sodium phosphate monobasic and Q2 = 0.2 M sodium phosphate dibasic) and 10 ml of sample were loaded onto a 5 ml Bio-Scale Mini CHT Type II column pre-equilibrated with 50 mM sodium phosphate pH 7. Protein was eluted with a 20 CV linear gradient from 0–50% B and 1 ml fractions were collected. ENrich™ SEC 70 Chromatography The pooled CHT fractions were concentrated to 200 μl (~20-fold) using a Millipore 5k concentrator. The sample was then injected via static loop onto an ENrich SEC 70 size exclusion column pre-equilibrated with 50 mM sodium phosphate and 150 mM NaCl (15% B). Protein was eluted using isocratic flow for 1.25 CV. 1 ml fractions were collected and analyzed by SDS-PAGE. SDS-PAGE Analysis For all chromatography scouting, samples were analyzed by SDS-PAGE on Any kD™ Criterion™ TGX Stain-Free™ polyacrylamide gels. Gels were run for 30 min at 300 mA/gel and then immediately imaged using the ChemiDoc™ imaging system. Purity Quotient Calculations Data for individual scouting runs were opened in the evaluation tab of the ChromLab software (Figure 2C). Peak integration was performed on the 280 and 525 nm traces. The table in the fractions tab containing peak area and peak relative area was imported into a Microsoft Excel spreadsheet. The purity quotient (PQ) for each wavelength was calculated by dividing the relative area (%) by the collected volume (ml). The purity quotient difference for prancer purple (PQDPP) was calculated for each fraction as PQ525 – PQ280 and graphed as a histogram. Histograms of the runs for each scouting type (column, pH, and %B) were graphed together for analysis. PQD equations: PQ = Relative Area (%)/Collected Volume (ml) PQPOI – PQcontaminant = PQD PQD > 0: more of the protein of interest (POI) than contaminants PQD < 0: more contaminants than POI PQD = 0: POI is present in equal amount as contaminants Prancer Purple Binding % Calculations Individual column scouting runs were opened in the evaluation tab of the ChromLab software. Peak integration using default settings was performed on the 525 nm trace. Switching to the manual integration tab, the peak list for each run was reduced to three peaks (flowthrough, elution, and 100% B) with the following start and end volumes: flowthrough 0.98–7.39 ml, elution 22.17–31.82 ml, and strip 45.49–52.41 ml. %B Scouting Identifies Optimal Elution Buffer Concentration and Gradient Once an optimal pH was established for prancer purple binding, we optimized 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 optimized 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 contaminants, 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).
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