Expression and Purification of Recombinant Protein to Generate a Monoclonal Antibody to the PX Domain of Tks5 α Isoform in Cancer Cells
An undergraduate honors thesis submitted in partial fulfillment of the requirements for the degree of Bachelor of Science in University Honors and Micro/Molecular Biology
Tyrosine kinase Src substrates, Tks4 and Tks5, are involved in the formation of podosomes and invadopodia during cell migration and cancer cell metastasis. Most recently, the α form of Tks5 has been discovered highly expressed in cancer cells, suggesting that the Tks5 α isoform plays essential roles in the regulation of invadopodia. In the Tks protein family, only the Tks5 α isoform contains a PX domain, which binds an important second messenger, PI (3,4) P2. Using a monoclonal antibody as an antigen-specific-probe to detect the protein of interest could be a useful tool that would provide a highly specific recognition at the molecular level. This project aims to develop a highly specific rabbit monoclonal antibody (mAb) to target the human Tks5 α isoform (specifically the PX domain) in cancer cells. In this study we describe the first step of the entire process, immunogen preparation. The results showed that the recombinant protein, hTks5 α isoform PX domain, was successfully expressed in BL21 E.coli competent cells and the purified antibody has high immunoreactivity. Other promising results obtained from western blot and immunofluorescence analyses indicated the developing antibody samples have high targeting effects on human Tks5 α PX domain.