Cloning And Application Of A Novel SPA From Staphylococcus Aureus (ATCC6538)

Because its immunological activities and target specificity, Antibody drugs and related reagents become the fast growing categories among the biological products. In the last decade the sale of the antibody drugs rose from $301 million to $25.8 billion.The propotion of the antibody drugs in the whole biological products is also risen from 1/5 to 1/3. Antibodies not only provide important tools in modern research of the life sciences, but also play an essential role in studies of structure and function of genes and proteins. Antibody purification, as an important step in production of antibody drugs and reagents, is critical for antibody industries. Affinity chromotography is key in antibody purification. The expensive price of the affinity chromtography products increases the cost of antibody drugs.Staphylococcus aureus Protein A (SPA) is a bacterial protein, which was found to bind Fc of of immunoglobulin G of some mammales in the 1970s. Subsequently, SPA is studied as a efficient affinity ligand for antibody purification. Presently, SPA is the most useful affinity ligand of affinity chromatograph. So, it is the very urgent task to improve the activity and stability of SPA.The genomic DNA of Staphylococcus aureus (ATCC6538) was used as the template for cloning SPA gene by PCR. The sequence result showed that this SPA gene was 1503bp in length, coding for 501 amino acids. Amino acid Alignment between Staphylococcus aureus (ATCC6538) and Staphylococcus aureus (CowanI) showed that the IgG binding domain of two SPA genes are completely identical, but the X regions were diffirences. So we published the SPA genes sequence in Genbank. The accession number was EU695225.Two efficient expression vectors, pHis-SUMO-SPA and pHis-SUMO-FSPA, were constructed. The two vectors were transformed into the host bacterium Rosetta, the full-length SPA protein and functional fragment were fused with SUMO tag. The SUMO tag enhanced the stability and activity of SPA proteins. After induced by IPTG, the expression condition, IPTG concentrations, times and tempretures, were optimized. The two fusion proteins were purified with AKTA purification system, and high-puity fusion proteins were harvested.The IgG binding ability and the stability of the recombinant SPA were tested by ELISA method. The results showed that the SPA with SUMO tag had the same IgG binding ability but better stability than SPA without SUMO tag. After coupled with the CNBr activated agarose, the IgG from rabbit serum could be purified by the SUMO-FSPA protein. These results indicated that SUMO tag could improve the stability of SPA and the fusion protein could be used as a model to develop new antibody purification products.