Cation Exchange Chromatography: A Technique to Isolate Target Recombinant Protein from Insect Cell Lysate Based on Net Surface Charge
Published: April 30, 2023
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 <…
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</str…
開示
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 |
