FAP1 is a cytoplasmic protein of <i>Saccharomyces cerevisiae</i> named for its association with FKBP12, one of four members of the immunophilin FK506 binding proteins (FKBPs) superfamily, said to be involved in protein folding. FAP1 has been found to confer resistance against the immunosuppressive, anticancer drug, Rapamycin. The action played by Ramapycin is because it competes for the FKBP12’s binding site in a mutually exclusive interaction. The FKBP12:rapamycin dimeric complex inhibits the TOR1 and TOR2 gene products which are part of TORC1, thus affecting regulation of cell growth and stress-responsive transcription factors. Due to its homology with human transcription factor NF-X1, and its rapamycin-induced nuclear translocation, we hypothesized that FAP1 could also function as a transcription factor in yeast. However, aside from its cytoplasmic position and affinity for FKBP12, little is known about its mode of action. Considering this protein’s mysterious nature, we aim to express and isolate rFAP1 (short) to study its interactions with other proteins. For this purpose, a 6xHis tag will be cloned in-frame to pET21aLIC:FAP1(s), a recombinant vector made previously, and then, to express it in the <i>E. coli </i>strain BL-21C43(DE3)pLysS+. Afterwards, the recombinant fusion protein will be isolated through IMAC (Ni-NTA) prior to generate an in-house antibody for further studies.

Expression and isolation of 6xHis-Tagged rFAP1(s) using immobilized metal affinity chromatography (IMAC)

Macromolecular Chemistry: The Second Century

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ACS Spring 2020 National Meeting & Expo

The official archive and repository for all ACS Meetings & Expositions abstracts and select posters or presentations.