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 <…

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…

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