Cation Exchange Chromatography: A Technique to Isolate Target Recombinant Protein from Insect Cell Lysate Based on Net Surface Charge

Overview

This video demonstrates the cation exchange chromatography technique to isolate recombinant protein from the insect cell protein lysate.

Protocol

1. Protein purification

1. 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

2. 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.
3. 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.
4. 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.
5. 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.
6. 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.
7. 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).
8. 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.

Materials

Name	Company	Catalog Number	Comments
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 7x14 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