Protein A is derived from a strain of Staphylococcus aureus and contains ﬁve regions that bind to the Fc region of IgG. As an afﬁnity ligand, protein A is coupled to Sepharose so that these regions are free to bind IgG. One molecule of protein A can bind at least two molecules of IgG.
Both protein A and a recombinant protein A are available from Cytiva. These molecules share similar speciﬁcities for the Fc region of IgG, but the recombinant protein A has been engineered to include a C-terminal cysteine that enables a single-point coupling to Sepharose. Single point coupling often results in an enhanced binding capacity.
The binding strength of protein A for IgG depends on the source species of the immunoglobulin as well as the subclass of IgG (Table 2). The dynamic binding capacity depends on the binding strength and also on several other factors, such as ﬂow rate during sample application.
Although IgG is the major reactive human immunoglobulin, some other types have also been demonstrated to bind to protein A. Interaction takes place with human colostral IgA as well as human myeloma IgA2 but not IgA1. Some human monoclonal IgMs and some IgMs from normal and macroglobulinaemic sera can bind to protein A.
Leakage of ligands from an afﬁnity medium is always a possibility, especially if harsh elution conditions are used. The multi-point attachment of protein A to Sepharose results in very low leakage levels over a wide range of elution conditions.
* Protein A Sepharose 4 Fast Flow and rProtein A Sepharose Fast Flow have a higher binding capacity, a more rigid matrix and provide more convenient alternatives to Protein A Sepharose CL-4B, which must be rehydrated before column packing.
** 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.
Figure 17 shows the puriﬁcation of mouse IgG2b from ascites ﬂuid on HiTrap® rProtein A FF 1 mL column using a syringe. The eluted pool contained 1 mg IgG2b and the silver stained SDS-PAGE gel conﬁrmed a purity level of over 95%.
Figure 17.Purifcation of mouse IgG2b from ascites on HiTrap® rProtein A FF 1 mL column using a syringe.
Figure 18 shows a larger scale puriﬁcation of monoclonal mouse IgG2a from a clariﬁed hybridoma cell culture on rProtein A Sepharose Fast Flow. Sample loading was over 9 mg IgG/mL of medium, with a 95% recovery of highly puriﬁed antibody.
Figure 18a.Puriﬁcation of a monoclonal IgG2a from clariﬁed cell culture on rProtein A Sepharose 4 Fast Flow.
Figure 18b.SDS-PAGE of starting material (lane 2) and eluate (lane 3). The samples were concentrated 10 times and reduced. Lane 1 and 4 are LMW markers. PhastSystem, PhastGel Gradient 10–15.
Column: HiTrap® Protein A HP, 1 mL or 5 mL, or HiTrap® rProtein A FF, 1 mL or 5 mL
Recommended ﬂow rates: 1 mL/min (1 mL columns) or 5 mL/min (5 mL columns)
Binding buffer: 0.02 M sodium phosphate, pH 7.0
Elution buffer: 0.1 M citric acid, pH 3–6
Neutralization buffer: 1 M Tris-HCl, pH 9.0
Centrifuge samples (10 000 g for 10 minutes) to remove cells and debris. Filter through a 0.45 µm ﬁlter. If needed, adjust sample conditions to the pH and ionic strength of the binding buffer either by buffer exchange on a desalting column (page 133, Buffer exchange and desalting for affinity chromatography) or by dilution and pH adjustment.
A HiTrap® column can be used with a syringe, a peristaltic pump or connected to a liquid chromatography system, such as ÄKTAprime plus.
* Since elution conditions are quite harsh, 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.
Table 3 gives examples of some typical binding and elution conditions that have been used with Protein A Sepharose.
Binding strengths are tested with free protein A. They can be used as a guide to predict the binding behaviour to a protein A afﬁnity medium. However, when coupled to an afﬁnity matrix the interaction may be altered. For example, rat IgG1 does not bind to protein A, but does bind to Protein A Sepharose.
IgGs from most species and subclasses bind protein A near to physiological pH and ionic strength. Avoid excessive washing if the interaction between the protein of interest and the ligand is weak, since this may decrease the yield.
With some antibodies, such as mouse IgG1, it might be necessary to add sodium chloride up to 3 M in the binding buffer to achieve efﬁcient binding when using protein A, for example 1.5 M glycine, 3 M NaCl, pH 8.9.
Alternative elution buffers include: 1 M acetic acid, pH 3.0 or 0.1 M glycine-HCl, pH 3.0 or 3 M potassium isothiocyanate.
Potassium isothiocyanate can severely affect structure and immunological activity.
Use a mild elution method when labile antibodies are isolated. Reverse the ﬂow of the wash buffer and elute with 0.1 M glycyltyrosine in 2 M NaCl, pH 7.0 at room temperature, applied in pulses. (Note: glycyltyrosine absorbs strongly at wavelengths used for detecting proteins). The speciﬁc elution is so mild that the puriﬁed IgG is unlikely to be denatured.
To increase capacity, connect several HiTrap® Protein A HP or HiTrap® rProtein A FF columns (1 mL or 5 mL) in series or pack a larger column with Protein A Sepharose 4 Fast Flow or rProtein A Sepharose 4 Fast Flow (Appendix 3, Column packing and preparation for affinity chromatography).
Desalt and/or transfer puriﬁed IgG fractions into a suitable buffer using a desalting column (see page 133, Buffer exchange and desalting for affinity chromatography).
Reuse of Protein A Sepharose and rProtein A Sepharose media depends on the nature of the sample and should only be considered when processing identical samples to avoid cross-contamination.
* 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. Short term refers to the pH interval for regeneration, cleaning-i lace and sanitization procedures.
** Protein A Sepharose 4 Fast Flow and rProtein A Sepharose 4 Fast Flow have a higher binding capacity, a more rigid matrix and provide more convenient alternatives to Protein A Sepharose CL-4B which must be rehydrated before column packing.
These media and columns tolerate high concentrations of urea, guanidine HCl and chaotropic agents.
Wash media and columns with 20% ethanol (use approximately 5 column volumes for packed media) and store at +4 to +8 °C.