Conversion of the C-Terminus of Transcription Factor Pdx1 to a Vehicle for Small Peptide Investigations

Open Access
- Author:
- Olson, Jarod
- Area of Honors:
- Chemistry
- Degree:
- Bachelor of Science
- Document Type:
- Thesis
- Thesis Supervisors:
- Scott A Showalter, Thesis Supervisor
Elizabeth A Elacqua, Thesis Honors Advisor
Benjamin James Lear, Faculty Reader - Keywords:
- biophysical
Pdx1
centFCP1
IDP
intrinsically disordered protein
fluorescence anisotropy
NMR
monte carlo
small peptides
q5 mutagenesis - Abstract:
- Biophysical analysis of small fragments of larger proteins has proven useful in identifying the particular interactions and nuances of individual protein domains. However, Escherichia coli does not reliably express recombinant proteins smaller than four kDa, and the purchase of synthetic peptides is expensive. In order to produce these small fragments in a cost-effective manner, we have used mutagenesis to develop a pET49b(+) construct containing restriction sites for subcloning a sequence of interest. The insertion is embedded within the sequence of the C-terminal domain of pancreatic and duodenal homeobox 1 (Pdx1-C), an intrinsically disordered protein (IDP) that is stable and large enough to be reliably expressed. This construct, which we have termed Proteus, was engineered to contain protease recognition sites that permit post-expression isolation of the small protein via protease cleavage. This technique allows for the synthesis of small proteins or protein fragments. As a demonstration of the utility of Proteus, we have inserted a 3.8 kDa peptide fragment of interest to our lab, the central domain of FCP1 (centFCP1). We then expressed this new construct (centFCP1-Pdx1) to investigate centFCP1’s binding interaction with RAP74 via fluorescence anisotropy binding assays and NMR spectroscopy. Experimental biophysical characterization was complemented with orthogonal computational simulations for further study. Thus far, it has been demonstrated that the construct, Proteus, successfully allowed for subcloning of centFCP1 and subsequent expression of centFCP1-Pdx1. In addition, the techniques and assays mentioned have shown that the centFCP1-Pdx1 remains able to bind to RAP74, oligomerizes at some concentration above 10 μM, and indicates a tendency for centFCP1’s partial sampling of helical conformations. In future work, I will collect NMR spectra of centFCP1-Pdx1 at decreasing concentrations to more accurately determine the concentration at which the peptide begins to oligomerize. Lastly, I will use isothermal titration calorimetry to thermodynamically determine the binding strength of centFCP1-Pdx1 to RAP74 at concentrations below the oligomerization point.