Team:CU-Boulder/Design
Overall Idea and Structure of Protein
Our overall idea is to switch an antibody on and off. First we had to find an Fab unit that could easily be made in E.Coli and has a clear crystal structure on the protein database. We found the pembrolizumab Fab in complex with PD1 antigen. We also had to find a complex that could quickly and easily be activated and inactivated. This is why we decided to use the Rapamycin complex. Two vectors were needed to test our theory. First we needed a vector for the engineered Fab/Rapamycin complex protein. Another vector was needed to produce the PD1 antigen to test the engineered antibodies binding affinity.
Designing the Vectors
Vector 1
The first vector contained the engineered antibody. A long linker was used to connect both chains of the engineered antibody so that it could be purified as one protein. A GST(glutathione S-transferase) protein was added onto the c-terminus of the protein in order to make the protein more soluble and to purify the protein in a glutathione column.
Vector 2
The second vector contains the PD1 antigen. The PD1 antigen is also attached to a maltose binding protein(MBP) at the N-terminus that makes the protein more soluble. A his tag is attached at the C-terminus and will be used for protein purification in a nickel column.
Vector to Protein
Once we inserted our g-block into the pet-42a vector through Gibson Assembly we transfected E.Coli cells with our vector and grew up the transfected cells. Later the proteins will be purified using a glutathione column for the engineered protein and a nickel column. Because of the MBP, GST, and His tag we added onto our proteins they are able to be soluble and can bind to our columns to be purified.
Testing Our Design
Our on/off switch will be tested through an ELISA assay. In this assay our engineered protein will be fixed by the GST to a bead on a plate. The PD1(which has an attached marker to the MBP) will be washed over the plate with the engineered protein. For the rapamycin on switch PD1 should not bind the fab because rapamycin is not present. Then rapamycin will be washed over the plate and bind to the fab, activating the fab binding affinity. Then when PD1 is washed over the plate for a second time it should bind to the fab.