Anion-Exchange Chromatography-Based Protein Purification: A Separation Technique to Isolate a Protein of Interest From Dialyzed Bacterial Lysate Based on Net Charge

Overview

In this video, we demonstrate an anion-exchange chromatography technique to purify human calcium-binding protein S100A12 based on the net surface charge of the protein from an Escherichia coli culture.

Protocol

1. Protein Purification

1. Anion-exchange chromatography
   1. Dialysis
      1. Prepare anion-exchange chromatography (AIEX) buffer A by dissolving 20 mM Tris, 1 mM EDTA and 1 mM ethylene glycol-bis(2-aminoethylether)-N, N, N', N'-tetraacetic acid (EGTA) in deionized water and adjust the pH to 8.5 with HCl. For dialysis prepare 2 x5 L and for chromatography 2x 1 L of AIEX buffer A.
         NOTE: The dialysate volume should be at about 100 times the sample volume. All buffers used for chromatography should be filtered (0.45 μm or smaller) and degassed (e.g., by ultrasonic bath or vacuum degassing).
      2. Cut dialysis tubing (molecular weight cut-off [MWCO]: 3.5 kDa) into an appropriate length with additional space for air to ensure sample buoyancy above the rotating stir bar.
         NOTE: Glycerol preserves the membrane and must be removed before use.
      3. To reduce the viscosity of the cleared protein solution, dilute the solution with 25 mL of AIEX buffer A to facilitate subsequent application to the chromatography column. Attach the first closure onto the tubing, load the sample into the membrane and attach the second closure at least 1 cm from the top end of the tubing.
      4. Place the 5 L container with AIEX buffer A on a stir plate, add a stir bar and the membrane filled with protein solution. Adjust the speed to rotate the sample by avoiding interference with the rotating stir-bar. Dialyze for 12−24 h at 4 °C, then replace the dialysate buffer (AIEX buffer A) by a fresh pre-cooled preparation and continue for at least 4 additional hours. Transfer the dialyzed protein solution to a 50 mL tube and filter through a 0.45 μm filter unit.
         NOTE: Storage possible.
   2. Chromatography
      1. Start the liquid chromatography system (FPLC) with general maintenance, connect column buffers AIEX A and AIEX B (AIEX buffer A with 1 M NaCl) and the anion-exchange resin containing column. Refer to Table 1 for general chromatographic parameters.
         NOTE: Buffers, column and FPLC equipment should be equilibrated to the same temperature before starting the run (refer to chromatographic parameters in Table 1, Table 2).
      2. Equilibrate the column with AIEX buffer A, subsequently load the sample onto the column and elute the proteins with a linear gradient from 0% to 100% high-salt buffer (AIEX B). Refer to Table 2 for a detailed method protocol.
      3. Collect 2 mL fractions during elution and analyze 10 μL of each fraction on a Coomassie-stained 15% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Pool the fractions containing S100A12 protein for dialysis.
         NOTE: The molecular weight of S100A12 is 10,575 Da.

Representative Results

Table 1: Detailed information on the applied parameters of anion-exchange chromatography.

Bed Volume (CV)	75 mL
Monitor	Absorbance at 280 nM
Pressure Max	3 bar
Column buffer A	20 mM Tris-HCl, 1 mM EDTA, 1 mM EGTA, pH 8.5
Column buffer B	20 mM Tris-HCl, 1 mM EDTA, 1 mM EGTA, 1 M NaCl, pH 8.5
Sample Volume	variable
Flow Rate	1−2 mL/min
Temperature	4 °C

Table 2: Detailed information on the used method of anion-exchange chromatography.

Block	Volume	Buffer	Outlet
Equilibration	1−2 column volumes (CVs)	A	Waste
Sample load	n/a	A	Waste
Wash out unbound sample	1 CV	A	High volume outlet
Gradient–Elution	0−100 % Buffer B in 1 CV	A to B	Fraction collector
Wash out–Buffer B	1 CV	B	Waste
Re-Equilibration	2 CVs	A	Waste

Materials

Name	Company	Catalog Number	Comments
25% HCl	Carl Roth	X897.1
4−20% Mini-PROTEAN TGX Protein Gels	BioRad	4561093
Coomassie Briliant Blue R250 Destaining Solution	BioRad	1610438
Coomassie Briliant Blue R250 Staining Solution	BioRad	1610436
EDTA disodium salt dihydrate	Carl Roth	8043.1
EGTA	Carl Roth	3054.3
Q Sepharose Fast Flow	GE Healthcare	17-0510-01	Resin for anion-exchange chromatography
Sodium chloride (NaCl)	Carl Roth	3957.2
Sodium hydroxide	Carl Roth	P031.1
Tris Base	Carl Roth	4855.3
0.45 µm syringe filter	Merck	SLHA033SS
5 L measuring beaker	Carl Roth	CKN3.1
50 mL conical centrifuge tubes	Corning	430829
Dialysis Tubing Closures	Spectrum	132738
Fraction collector tubes 5 mL	Greiner	115101
Spectra/Por Dialysis Membrane (3.5 kDa)	Spectrum	132724
Steritop filter unit	Merck	SCGPT01RE
ÄKTA purifier UPC 10	GE Healthcare		FPLC System
Fraction collector	GE Healthcare	Frac-920
Mini Protean Tetra Cell	BioRad	1658000EDU
NanoPhotometer	Implen	P330
pH meter	Knick	765