Engineering and expresSION of polyprotein A

Abstract Protein A is a surface protein found in the cell wall of bacteria Staphylococcus aureus and is encoded by spa gene. It is extensively used in biomedical research because of its ability to bind to immunoglobulins with high affinity. The folding of five homologous Ig- binding protein domains leads to the composition of this three-helix bundle protein and each domain is capable of binding to many mammalian proteins, essentially IgG. IgG is a type of immunoglobulins (antibodies) circulating in the blood that aid in the phagocytic destruction of antigens. The objective of this project was to create octamers of the two, out of the five, subunits of this protein: subunit B4 and B5. Starting from the monomeric unit, we applied molecular biology protocols and engineered a poly protein A made from eight repeats. The entire project can broadly be classified into three major steps: monomer to dimer, dimer to tetramer, and tetramer to octamer. Each step further had sub steps that included digesting the fragment and vector, ligating them together, screening the ligation product, finally followed by sequencing to double check the obtained product. We have finally expressed this protein and tested its antibody activity using SDS-PAGE gels and other binding assays