An Affinity Chromatography Technique for the Purification of a Recombinant Bacterial Protein

Abstract

Source: Becker, B., et al. A Simple Fluorescence-based Reporter Assay to Identify Cellular Components Required for Ricin Toxin A Chain (RTA) Trafficking in Yeast. J. Vis. Exp. (2017).

This video demonstrates the purification of a polyhistidine-tagged recombinant protein using nickel-based affinity chromatography. It outlines key steps, including bacterial lysis by sonication, clarification and filtration of the lysate, and selective binding and elution of the target protein using imidazole.

Protocol

1. Purification of His-tagged RTA via Affinity Chromatography

1. Sonicate cells on ice using the following protocol: 15 s pulse (20 microns), 30 s pause. Repeat this step five times.
2. Centrifuge cell lysate at 21,000 x g and 4 °C for 15 min and filter supernatant using a sterile syringe filter system (0.2 µm pore size).
   NOTE: Cell pellets of successfully sonicated samples show transparent borders.
3. Use an automated purification system equipped with a 5 mL Ni²⁺-based affinity column to purify the His-tagged RTA fraction from the sterile-filtered E. coli supernatant. In general, use an elution speed of 1 mL/min and cool the whole purification system to prevent inefficient toxin binding and loss of toxin activity.
   NOTE: Parameters for efficient RTA purification are listed in Table 1.
   1. Briefly, equilibrate the affinity column with 20 mL of binding buffer to remove the storage buffer. Apply sterile-filtered supernatant onto the affinity column using a syringe.
   2. Wash the column with 25-35 mL of binding buffer to remove the unbound proteins from the column. Perform the washing step until the UV absorbance at 280 nm is close to the initial UV value.
   3. Elute bound RTA fraction in 20-35 mL of elution buffer (500 mM imidazole, 500 mM NaCl, 20 mM KH₂PO₄, pH = 7.2) and keep the sample on ice (Figure 1A and Figure 1B).
      NOTE: Elution of the RTA fraction is marked by an increase in UV absorption. Please note to exclusively collect this fraction to prevent contamination with nonspecific bound proteins.

Table 1: Parameters used for column-based affinity purification and desalting. List of all relevant column settings (e.g., flow rate, elution buffer concentration, equilibration and washing step duration) and measured parameters (e.g., conductance or UV absorption).

Affinity chromatography parameter
Measuring parameters	UV (280 nm), Elution buffer concentration, Conductance
Settings	Variable/Unit	Value
Flow rate	mL/min	1
Maximum column pressure	MPa	0.35
Wavelength	nm	280 nm
Binding buffer	Pump position	A
Elution buffer	Pump position	B
Starting concentration elution buffer	%	0
System volume compensation	mL	10
Column equilibration	mL	20
Sample injection over superloop/syringe	mL	cell lysate volume
Washing step	mL	20-35
Elution step	mL	20-35
Concentration elution buffer during elution	%	100
Recalibration of column with 20% EtOH	mL	50
Desalting parameter
Measuring parameters	UV (280 nm), Conductance
Settings	Variable/Unit	Value
Flow rate	mL/min	4
Maximum column pressure	MPa	0.35
Wavelength	nm	280 nm
Binding buffer	Pump position	A
Elution buffer	Pump position	B
Starting concentration elution buffer	%	0
System volume compensation	mL	10
Column equilibration	mL	20
Sample injection over superloop/syringe	mL	cell lysate volume
Desalting buffer injection (0.8 M sorbitol)	mL	100
Recalibration of column with 20% EtOH	mL	50

Results

Figure 1: Representative affinity chromatography and desalting results of E. coli

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Spectrophotometer Ultrospec 2100 pro	Amersham
Soniprep 150	MSE		old model, other models available
Fluoroskan Ascent	Thermo Scientific	5210470	old model, not available anymore
ÄKTAPurifier	Thermo Scientific	28406266	Product is discontinued and replaced
HisTRAP HP column	GE Healthcare	17-5248-02
HiTRAP desalting column	GE Healthcare	11-0003-29
Midisart sterile filter	Sartorius	16534K	0.2 µm pore size
BCA protein assay kit	Pierce	23225
660 nm assay kit	Thermo Scientific	22660
96 well plates	Thermo Scientific	260860