focus on Chromatography
Protein Purifi cation Workfl ow Development Using Bio-Rad’s NGC™ Chromatography System
Paul Ng, Kiran Kaur, Jonathan Kohn, Anna Quinlan, and Jeff Habel Bio-Rad Laboratories, Inc, 2000 Alfred Nobel Drive, Hercules, CA, USA
High purity protein is a common requirement for biochemical and structural studies. A common approach is to recombinantly express an affi nity-tagged version of the protein of interest. This is, however, not always a viable option. Here we discuss protein purifi cation workfl ow development for untagged proteins and introduce a new indicator of method performance, the purity quotient difference (PQD).
High purity protein samples are essential for applications from protein structure determination and biochemical characterisation to antibody production. A standard purifi cation approach is the use of affi nity tags such as hexahistidine (6x histidine) or glutathione-S-transferase (GST) tags. These tags increase the throughput and effi ciency of the protein purifi cation workfl ow, as protocols are often readily available or supplied by the manufacturer.
Affi nity tagging is not always a viable option. Some proteins are unstable or inactive once tagged or require posttranslational modifi cations that do not permit recombinant expression. In these cases, researchers often settle for lower purity protein rather than exhaustively explore purifi cation options, since the purifi cation optimisation process can be time and labour intensive when no particular column resins or buffer conditions are dictated by an affi nity tag.
Whether purifying tagged or untagged proteins, an optimal purifi cation workfl ow includes column, pH, and elution buffer gradient (%B) optimisation for each purifi cation step (Figure 1). Here we show that the Bio-Rad NGC medium pressure chromatography system, equipped with column switching valve, sample pump, buffer blending valve, and the ChromLab™ software scouting feature, allows us to automate the process of column, pH, and %B optimisation. 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 fi ndings cement the importance of column, pH, and buffer scouting when developing purifi cation workfl ows 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.
Materials and Methods Protein Expression
After overnight induction, cells were harvested by centrifugation with a Sorvall SA-600 rotor at 10,000 rpm (14,500 RCF) for 10 min. Kanamycin was used at a concentration of 50 µg/ml for liquid and solid phase growth.
Cell Lysis
Cell pellets were resuspended in 20 ml B-Per (Pierce Cat #78243) lysis buffer supplemented with 20 µl of 1 mg/ml DNase I and 200 µl of 1 M MgCl2
. Lysates were clarifi ed by centrifugation at 10,000 rpm (14,500 RCF) for 10 min.
Column Scouting Bio-Rad’s 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. Clarifi ed 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 purifi cation steps were carried out at a 1 ml/min fl ow rate. Fractions from each run were analysed by SDS-PAGE.
pH Scouting
Once the Foresight Nuvia Q column was selected as the fi rst 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 analysed by SDS-PAGE.
Figure 1. Protein purifi cation workfl ow scouting.
The kanamycin-resistant prancer purple expression vector (DNA 2.0) was transformed into HB101
E.coli using the standard heat shock method. A single colony was picked into 10 ml LB supplemented with kanamycin (LB/kan) and grown overnight at 37°C. Cells were then pelleted using an Eppendorf 5804 centrifuge (rotor A-4-44) at 4,000 rpm (2,880 RCF) for 10 min, resuspended in 10 ml of fresh LB/kan, and inoculated into 1 L of LB/kan. Cultures were grown at 37°C on a shaker set to 200 rpm. Prancer purple expression was induced when cultures reached an OD600
of 0.4, by adding IPTG to a fi nal concentration of 0.1 mM.
LAB ASIA - MARCH/APRIL 2014
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