Affinity Chromatography - Department of Molecular and Cellular ...

Protein A fusion proteins
IgG Sepharose 6 Fast Flow
Recombinant fusion proteins containing a protein A tail and protein A can be purified on
IgG Sepharose 6 Fast Flow.
Purification option
Product Binding capacity/ml medium Maximum operating flow
IgG Sepharose Fast Flow 2 mg protein A at pH 7.5 400 cm/h*
*See Appendix 4 to convert linear flow (cm/h) to volumetric flow rate. Maximum operating flow is calculated from
measurement in a packed column with a bed height of 10 cm and i.d. of 5 cm.
Purification example
Figure 28 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.
A) Sample: Bacterial suspension containing ZZ-IGF-1 fusion protein,
automatically sampled from fermentation broth, 500 µl
Column: IgG Sepharose 6 Fast Flow (0.5 x 2.5 cm)
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
A
A280 nm
0.1
Binding
buffer
Wash
buffer
Elution
buffer
B
A600 nm
120
100
80
Conc.
A600
Conc. (mg/ml)
600
500
400
60
300
0.05
ZZ-IGF-1
40
200
20
100
0
10.0
Time (min)
0
10 20 30
0
40 50
Time (h)
Fig. 28. 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 A 600 nm
.
Performing a separation
Binding buffer: 0.05 M Tris-HCl, 0.15 M NaCl, 0.05% Tween 20, pH 7.6
Wash buffer: 5 mM ammonium acetate, pH 5.0
Elution buffer:
0.5 M acetic acid, adjusted to pH 3.4 with ammonium acetate
Neutralization buffer: 1 M Tris-HCl, pH 9.0
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