Discipline: Biological Sciences
Subcategory: Cancer Research
Wendy Anyona - Delaware State University
Co-Author(s): Karl E. Miletti, Delaware State University, Dover, DE
We have previously tagged the CD44 intracytoplasmic domain (CD44-ICD) with the green fluorescent protein (GFP), and used this fluorescent fusion protein in imaging studies as well as for chromatin immunoprecipitation (ChIP) assays (Miletti-Gonzalez et al., 2005). GFP has been widely used as a fluorescent tag for many different proteins. The GFP length of 238 aminoacids (aa) makes it an ideal tag for a typical eukaryotic protein size (~400 aa) or larger. However, for smaller proteins or polypeptides, the use of this tag is controversial due to the addition of a domain that sometimes is even larger than the original untagged protein. This is the case of the CD44-ICD, which with only 74 aa in length, is about three times smaller than the relatively large GFP protein (238 aa, ~25 kDa). Therefore, the generation of a CD44-ICD tagged with a small fluorescent protein is a wanted improvement. For that purpose we decided to use the iLOV fluorescent protein (Chapman et al., 2008). The LOV (light, oxygen or voltage) domain, derived from a plant blue light receptor is about half size of GFP (~11 kDa) and with 111 aa long is not much larger than the CD44-ICD. Making use of a considerable large multi-cloning site in a commercially available plasmid carrying the iLOV domain, we will test three different ‘connecting bridge’ lengths between the iLOV domain and the CD44-ICD. We have already PCR amplified and purified the three CD44-ICD inserts, which we expect to clone in the iLOV plasmid by homologous recombination. Ultimately, we expect to identify an iLOV/CD44-ICD fusion protein with enhanced imaging capabilities, which in part will help us validate the GFP/CD44-ICD fusion protein data.
Funder Acknowledgement(s): This project was funded by NSF HBCU-UP RIA, Grant No.1700228 and Delaware Economic Development Office, Grant No.103
Faculty Advisor: Karl E. Miletti, firstname.lastname@example.org
Role: Using PCR amplification, I was able to tag the CD44-ICD with the iLOV plasmid. Which allowed me to clone the CD44-ICD by homologous recombination. We made our fusion protein to be able to follow the CD44-ICD movement in the cell. That should start, once the CD44 protein is cut and the ICD is made and goes into the nucleus and interacts, if at any interaction at all.