Technical Articles

The development of Protein A Sepharose with high binding affinity and mild elution condition Time:9/24/2009 12:15:11 AM Count:
The development of Protein A Sepharose with high binding affinity and mild elution condition
Protein A, a bacterial cell wall protein isolated from Staphylococcus aureus, binds to mammalian immunoglobulins (IgGs) mainly through Fc regions of antibodies. It can be cooperatively coupled to the matrix of Sepharose, and such modified sepharose can be used for antibody purification.
At the beginning, the coupled protein A was from natural resources. Native protein A contains five IgG binding domains and some other domains of unknown functions. The molecular weight is about 42KD (Figure 1). The protein A-coupled sepharose beads have strong affinity to IgG and demonstrate a large binding capacity. However, the containing of some domains of unknown functions has led to the unspecific binding of protein A with other type of proteins. As a result, the protein A-coupled sepharose beads have limited ability in getting highly purified IgG.


Figure 1. Structure of Native Protein A

In order to reduce the unspecific binding, a new generation of protein A was produced by cloning the five domains with specific binding to IgG and recombining them into one protein (recombinant protein A). In this way, the IgG non-specific binding domains were removed. This recombinant protein A (rProtein A) sepharose beads perform much better purification efficiency and effect, therefore, have been widely used in lab and industry since they are developed. However, a major drawback of rProtein A is that a low pH elution buffer is often required to completely elute a protein of interest, while a low pH usually cause denaturation of some proteins. This is because the IgG binding domains (E, D, A, B, C) of rprotein A have different binding affinity with Fc of IgG, so that the elution condition is not consistant and low pH is required. With this regards, it is believed a mild elution condition can be achieved by recombining less number of IgG-binding domains with the same affinity into one protein.

Putus recently developed an advanced type of rprotein A sepharose beads by introducing three IgG-binding domain B into the protein, as shown in Figure 2. The rProtein sepharose beads from both Putus and GE were used to purify human plasma, and the result of purification was evaluated by SDS-PAGE, as shown in Figure 3.   



     Figure 2. Structure of rProtein A.  GE:  rProtein A Sepharose 6 FF from GE Company, containing 5 binding domains of E, D, A, B, C with the ratio of 1:1:1:1:1; Putus: rPtrotein A Sepharose 6 FF from Putus, containing 3 domains of B.



Figure 3. SDS-PAGE results of purification of human plasma IgG using GE and Putus rProtein A sepharose beads. The purificactions were carried out according to instruction manual of GE and Putus, respectively. The elution buffers¡¯ pH for lane 2 and 3 are 3 and 4.5, respectively. Lane 1: MW standards; Lane 2: human plasma purified by GE rProtein A Sepharose; Lane 3: human plasma purified by  Putus rProtein A Sepharose; Lane 4: human plasma.

As shown in lane 2 and 3, the IgG with high purity is obtained by both GE and Putus'rProtein A Sepharose. But the elution condition of lane 3 is milder than that of lane 2. The result has indicated that Putus'rProtein A Sepharose, containing 3-isotype's binding domain, have same purification effect with compare to GE's product, meanwhile, the purification using Putus'rProtein A sepharose beads can be performed under milder condition so that the aggregation and denaturation of proteins to be purified can be reduced.