5 %B Scouting
After pH 7.5 was selected, the scout feature was used to optimise %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 analysed 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 analysed by SDS-PAGE.
SDS-PAGE Analysis
For all chromatography scouting, samples were analysed 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
as a histogram. Histograms of the runs for each scouting type (column, pH, and %B) were graphed together for analysis.
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. 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
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
Figure 2. Column scouting. A,
Prancer Purple Binding % Calculations
Individual column scouting runs were opened in the evaluation tab of the ChromLab software. Peak integration with 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.
Results Column Scouting Identifies Optimal Resin for Protein Binding
Untagged prancer purple was expressed recombinantly in
E.coli. Prancer purple has a predicted isoelectric point (pI) of 6.65 and the chosen lysis buffer a pH of 7.5, making anion exchange an excellent first step in our purification workflow. Bio-Rad has a number of different anion exchange resins of various bead sizes and chemistries. We chose a larger bead size that could accommodate faster flow rates with our highly viscous crude lysate sample.
Three 1 ml anion exchange chromatography columns, Foresight Nuvia Q, Bio-Scale Mini UNOsphere Q, and Bio-Scale Mini Macro-Prep High Q, were attached to different ports of
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