Purification of Protein A-Tagged Proteins

Chapter 8, Extracted from Affinity Chromatography Vol. 2: Tagged Proteins, GE Healthcare, 2016

Recombinant tagged proteins containing a protein A tag can be purified on IgG Sepharose^® 6 Fast Flow. The AC medium is based on the Sepharose^® 6 Fast Flow matrix, with human IgG covalently coupled to it. The mechanical characteristics of this Fast Flow medium allows high flow velocities (up to 400 cm/h) to be used for rapid and convenient single-step purification of protein A-tagged protein conjugates produced in prokaryotic expression systems. Up to 2 mg/ml protein A can be bound to the ligand at pH 7.5.

IgG Sepharose^® 6 Fast Flow is also suitable for tandem affinity purification (TAP) of protein complexes, see the GE handbook Purifying Challenging Proteins: Principles and Methods, 28909531.

An alternative eluent is 0.1 M glycine-HCl, pH 3.0. Chaotropic agents may also be used for elution.

Characteristics of IgG Sepharose^® 4 Fast Flow are shown in Table 8.1.

Table 8.1. Characteristics of IgG Sepharose® 6 Fast Flow

Ligand	Composition	pH stability*	Particle size
Human polyclonal IgG	IgG coupled to Sepharose® Fast Flow by the cyanogen bromide method.	Short term 3–10 Long term 3–10	90 µm

* Short term refers to the pH interval for regeneration, cleaning-in-place and sanitization procedures. Long term refers to the pH interval over which the medium is stable over a long period of time without adverse effects on its subsequent chromatographic performance.

Performing a separation using IgG Sepharose^® 6 Fast Flow

Buffer Preparation

Binding buffer: Wash buffer: Elution buffer: Neutralization buffer:

0.05 M Tris-HCl, 0.15 M NaCl, 0.05% Tween 20, pH 7.6 5 mM ammonium acetate, pH 5.0 0.5 M acetic acid, adjusted to pH 3.4 with ammonium acetate 1 M Tris-HCl, pH 9.0

Purification

1. Pack the column (see Appendix 3, Characteristics of Dextrin Sepharose High Performance products) and wash with at least 5 column volumes of binding buffer.
2. Equilibrate the column with approximately 5 column volumes of binding buffer.
3. Wash with 2–3 column volumes of acetic acid followed by 5 column volumes of binding buffer.
4. Apply the sample.
5. Wash with 10 column volumes binding buffer.
6. Wash with 2 column volumes of wash buffer or until no material appears in the eluent (determined by UV absorbance at A280 nm).
7. Elute with 2–5 column volumes of elution buffer.*
8. Immediately re-equilibrate the column with binding buffer until the eluent reaches pH 7.0 (the IgG may denature if left at a lower pH).

* Since elution conditions are quite harsh, it is recommended to collect fractions into neutralization buffer (60 µL – 200 µL 1 M Tris-HCl, pH 9.0 per ml fraction), so that the final pH of the fractions will be approximately neutral.
This method, while giving a concentrated eluate, can only be used if the fusion product is stable under the acid conditions.

Application example

Figure 8.1 shows automatic on-line monitoring of the production of a secreted fusion protein during fermentation. The fusion protein, ZZ-IGF-1 is insulin-like growth factor 1 fused with a derivative of protein A (designated ZZ), expressed in E. coli.

Column:	IgG Sepharose® 6 Fast Flow (0.5 × 2.5 cm)
Sample:	Bacterial suspension containing ZZ-IGF-1 fusion protein, automatically sampled from fermentation broth, 500 µl
Binding buffer:	0.05 M Tris-HCl, 0.05% Tween 20, pH 7.6
Wash buffer:	10 mM ammonium acetate, pH 4.6
Elution buffer:	0.2 M acetic acid, pH 3.2

Fig 8.1. (A) Chromatogram of a sample taken at one time point during fermentation. (B) Results from automatic monitoring of the product concentration during fermentation. Concentration of ZZ-IGF-1 is determined by integration of the ZZ-IGF-1 peak obtained during each chromatographic analysis. Bacterial density is measured manually at A600 nm.

Chemical stability

Avoid reducing agents such as 2-mercaptoethanol or DTT since they may disrupt disulfide bonds within the IgG ligand.

Storage

Wash with 5 column volumes of 20% ethanol at neutral pH and store at +4 °C to +8 °C.