Cation Exchange Chromatography: A Technique to Isolate Target Recombinant Protein from Insect Cell Lysate Based on Net Surface Charge
Abstract
Source: Agamasu, C. et al. Fully Processed Recombinant KRAS4b: Isolating and Characterizing the Farnesylated and Methylated Protein. J. Vis. Exp. (2020)
This video demonstrates the cation exchange chromatography technique to isolate recombinant protein from the insect cell protein lysate.
Protocol
1. Protein purification
- Prepare buffers A–H, as seen in Table 1.
| Buffer solution | Buffering agent (all 20 mM) | pH | NaCl (mM) | Imidazole (mM) | MgCl2 | TCEP |
| A | HEPES | 7.3 | 300 | – | 5 | 1 |
| B | HEPES | 7.3 | 300 | 35 | 5 | 1 |
| C | HEPES | 7.3 | 300 | 500 | 5 | 1 |
| D | MES | 6.0 | 200 | – | 5 | 1 |
| E | MES | 6.0 | – | – | 5 | 1 |
| F | MES | 6.0 | 100 | – | 5 | 1 |
| G | MES | 6.0 | 1000 | – | 5 | 1 |
| H | HEPES | 7.3 | 300 | – | 1 | 1 |
- Prepare the purification materials (see Table of Materials): protease inhibitor cocktail without EDTA or other chelators; immobilized metal affinity chromatography (IMAC) column; cation exchange chromatography (CEX) column; 0.45 μm syringe filter; 2 M imidazole (pH = 7.5); ultracentrifuge capable of 100,000 x g; high speed benchtop centrifuge capable of 4,000 x g; centrifugal filter units (10 KDa NMWL); spectrophotometer capable of reading at 280 nm; His6-tobacco etch virus (TEV) protease; and chromatography instrumentation capable of mixing two buffer solutions, applying solutions to columns, creating gradient elutions from column, and collecting fractions from columns.
- Remove the insect cell lysate from dialysis and centrifuge at 4,000 x g for 10 min to remove any precipitate. The final dialyzed, clarified sample at this point is still often hazy but can be applied without further processing onto the subsequent CEX column.
- Prepare a 20 mL cation exchange column (see Table of Materials) by washing with three CVs of Buffer G, then with three CVs of Buffer F.
NOTE: While we have not meticulously evaluated other resins, several colleagues have found poor resolution with resins other than SP Sepharose High Performance resin. - Dilute 20 mL of the dialyzed sample to a final NaCl concentration of 100 mM by adding 20 mL of Buffer E and apply the diluted sample to the cation exchange column.
- Continue to load the column by adding freshly diluted samples prepared as described above to the load tube as the previous dilution is nearing the end of the load.
NOTE: Diluting the entire sample at once to 100 mM NaCl has resulted in significant loss of target protein due to precipitation. - Wash the column to baseline Abs280 with Buffer F. This typically requires 3 CV. The protein is eluted from the column during a 400 mL (20 CV) gradient from Buffer F to 65% Buffer G, collected in 6 mL fractions (Figure 1B).
- Continue washing the column for an additional 1.5 CV (65% Buffer G) once the gradient is completed.
Representative Results
Figure 1: SDS-PAGE gels of the purification process. (A) IMAC capture from lysate. FNTA/B are the dark bands migrating at ~48 kDa and the His6-MBP-tev-KRAS4b is the dark band migrating at ~67 kDa. M = protein molecular weight ladder; T = total protein; L = clarified lysate/column load; F = column flow through. Fractions are eluted with an increasing imidazole concentration gradient. (B) CEX fractions labelled 1–5 correspond to the peak fractions from the elution peaks. (C) TEV digestion and second IMAC. C = pool from CEX step pre-TEV cleavage; L = load sample post-TEV cleavage. The species of interest are labeled. (D) One and five micrograms of final KRAS4b-FMe protein. The gels depicted are representative results from multiple productions.
Divulgazioni
The authors have nothing to disclose.
Materials
| 1.8 mL Safe-Lock Tubes, Natural | Eppendorf | 22363204 | |
| 11 mm Cl SS Interlocked Insert Autosampler Vials | Thermo Scientific | 30211SS-1232 | |
| 5427R Centrifuge | Eppendor | ||
| Acetonitrile, HPLC Grade | Fisher Chemical | A998-1 1L | |
| Cation Exchange Chromatography (CEX) column | GE Healthcare Life Sciences | 29018183 | HiPrep SP Sepharose High Performance |
| Exactive Plus EMR Mass Spectrometer | Thermo Scientific | ||
| Gilson vials 7×14 mm Tubes | GE Healthcare | BR-1002-12 | |
| High speed/benchtop centrifuge | Thermo Fischer Scientific | 05-112-114D | Capable of up to 4,000 xg |
| His6-Tobacco Etch Virus (TEV) protease | Addgene | 92414 | Purified as per Raran-Kurussi et al. (2017) Removal of Affinity Tags with TEV Protease. In: Burgess Brown N. (eds) Heterologous Gene Expression in E.coli. Methods in Molecular Biology, vol 1586. Humana Press, New York, NY |
| Protease Inhibitor Cocktail without EDTA or other chelators | Millipore Sigma | P8849 | |
| Ammonium Acetate | Sigma-Aldrich | 09689-250g | |
| Argon gas | Airgas | ARUP | |
| Formic Acid | Sigma-Aldrich | F0507-500MI | Use Reagent Grade or better |
| NGC Chromatography System | BioRad | 78880002 | NGC QuestTM 100 Chromatography system |
| Lipid extruder set with holder | AVANTI POLAR LIPIDS | 610023 | |
| Liquid nitrogen | Airgas | NI-DEWAR | |
| Ultra-15 Centrifugal Filter Units, 10K NMWL | Millipore Sigma | UFC901008 | PES membrane |
| Ultracel 10K MWCO Ultra 0.5 mL Centrifuge Filters | Amicon | UFC501024 | |
| Ultracentrifuge | Beckman Coulter | Optima – L80K | capable of 100,000 xg |
| CHAPS | Sigma | C3023 |
