Engineering of a Polyprotein Construct to Test its Binding Activity

Mentor 1

Ionel Popa

Mentor 2

Annie Eis

Start Date

1-5-2020 12:00 AM

Description

Protein A, a surface protein, found in the cell wall of bacteria Staphylococcus aureus is encoded by the spa gene. Its ability to bind to antibodies with high affinity employs its heavy use in biomedical research. The folding of five homologous protein binding domains leads to the structure 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 subunits of this protein: B4 and B5. Starting from the monomeric unit, we employed cloning techniques 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. The sub steps under each step include digestion of the fragment and vector followed by their ligation. The octameric unit was sequenced at the end 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.

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May 1st, 12:00 AM

Engineering of a Polyprotein Construct to Test its Binding Activity

Protein A, a surface protein, found in the cell wall of bacteria Staphylococcus aureus is encoded by the spa gene. Its ability to bind to antibodies with high affinity employs its heavy use in biomedical research. The folding of five homologous protein binding domains leads to the structure 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 subunits of this protein: B4 and B5. Starting from the monomeric unit, we employed cloning techniques 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. The sub steps under each step include digestion of the fragment and vector followed by their ligation. The octameric unit was sequenced at the end 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.