Production, Characterization And Application Of Recombinant Staphylococcal Protein A

Protein A, a cell wall constituent in most strains of Staphylococcus aureus, is covalently bound to the peptidoglycan moiety of the cell wall. Staphylococcal protein A (SPA) is a single polypeptide chain without tryptophan. It is composed of five homologous Ig-binding domains that fold into a three-helix bundle.In the present study, recombinant Staphylococcus protein A (SPA) was successfully constructed and expressed with S. aureus genome as a template. After the conditions of expression were optimized, recombinant Staphylococcus protein A, the size of which is 30 kDa, was performed high expression. What's more, the 30 kDa variant and protein A with the size of 45 kDa, as well as the 25 kDa protein A with repeat units of immunoglobulin-specific binding protein were expressed successfully in E. coli.The characterization of protein A with different sizes was performed, such as acid resistance, alkali resistance and the tolerance to high temperature. The purification conditions of different proteins were determined, and the target protein with the purity of up to 97% was obtained after optimization. The 25 kDa protein A showed high tolerance to 0.25 M hydrochloric acid. When treated with 0.5 M HCl for 30 min, the 25 kDa protein A was only degradated slightly. Staphylococcus protein A with the size of 30 kDa was almost no degradation after treatment with 0.2 M HCl for 0.5～1 h. Besides, both the 30 kDa and the 45 kDa protein A presented high tolerance to 0.5 M of hydrochloric acid when treated with acid for 1 h.Staphylococcus protein A with the size of 25 kDa was no degradation after treatment with 0.5 M NaOH for 40 min. When the concentration of sodium hydroxide is no more than 0.2 M, the 30 kDa protein A was stable even if treated for 1 h. The 45 kDa protein A showed high tolerance to 0.25 M of sodium hydroxide.The 25 kDa Staphylococcus protein A was stable when treated in the range of 4～70℃for 6 h. Compared with the former, the 45 kDa Staphylococcus protein A was quite instable at room temperature. Staphylococcus protein A with a molecular weight of 30 kDa was easy to be degradated at 4℃and room temperature. The optimum conditions of epoxy-activation of sepharose were optimized through an orthogonal experiment:epichlorohydrin (ECH) concentration was 20% (v/v); the amount of NaOH was 1 ml with the concentration of 0.8 M; activation time was 2.5 h and the reaction temperature was 40℃. In addition, it was found that -SH could be coupled onto the epoxy-activated sepharose when pH was 6.4～8.0, while-NH2 could be coupled when pH was over 10. Rabbit immunoglobulin was purified to homogeneity using Protein A sepharose 4FF when the concentration of NaCl was 0.3 M.