Our goal was to build a hyper stable CPP-scFv(Ras) antibody that can remain functional inside the cell’s reducing environment and successfully bind to the Ras protein. The antibody inhibited the signaling of Ras protein, which could possibly be used as a cancer therapy. We demonstrated our first design-build-test cycle in four main stages: cloning, expression, purification and assay.
In the experiment, a CPP-scFv(Ras) DNA sequence designed with a His-6 polyhistidine tag was used to purify the target protein. Using PCR (Polymerase Chain Reaction) Amplification, the specific target DNA sequence was run through the PCR program, generating an ample amount of the target DNA of the scFv(Ras) antibody.
With the amplified DNA of the scFv (Ras), the sequence was inserted into a vector by LIC (Ligation Independent Cloning) methods. Once the DNA was cloned into a recombinant plas- mid, the vector was transformed into a DH5a strain of E. coli bacteria. Once a colony of E. coli cells grew, the DNA of the DH5a strain E. coli was extracted and again transformed into another E-coli competent cell (BL21(DE3)) which was suitable for the expression of proteins. After the induction, the E.coli competent cell (BL21(DE3)) colony with the plasmid for protein synthesis had expressed high amounts of scFv(Ras).
Affinity chromatography begun with a lysis buffer through the Ni-NTA resin for equilibra- tion then the cell lysate and the washing buffer was added to capture our target protein and wash away all other proteins in the protein soup. Lastly, an elution buffer with imidazole was added to the column so that the imidazole competed with the proteins bound to the Ni- NTA resin and eventually elute our target proteins.
Finally, through sodium dodecyl sulfate polyacrylamide electrophoresis (SDS-PAGE), the proteins were separated based on their molecular mass, confirming the existence of CPP-scFv(Ras).
The purified antibody was used for the binding assay. Human Ras is going to be used for the different assays.