Characterization of Elastin-like Polypeptide Fusion Proteins for the Development of Drug-Free Macromolecular Therapeutics

Armstrong, Blair1,2, Steinhauff Douglas1,2, Ghandehari, Hamid1,2,3 1 Department of Biomedical Engineering, University of Utah, Salt Lake City, UT, 36 S Wastach Dr 84112 2 Utah Center of Nanomedicine, Nano Institute of Utah, University of Utah, Salt Lake City, UT, 36 S Wastach Dr 84112 3 Department of Pharmaceutics and Pharmaceutical Chemistry, University of Utah, Salt Lake City, UT, 36 S Wastach Dr 84112

Advancement of diseases resistant to standard treatment has created the need for novel biomaterials to bridge current clinical gaps.  Drug-free macromolecular therapeutics (DFMTs) are one approach that have enabled pharmaceutical effects without the use of low molecular weight therapeutics. DFMTs function by clustering cell receptors to elicit a cellular response. Elastin-like polypeptides (ELPs) have been used as clustering agents due to their thermoresponsive behavior and can potentially cluster cell receptors at their lower critical solution temperature (LCST). An LCST is the temperature at which polymer-polymer interactions become more favorable than solvent-polymer interactions resulting in intra- and inter-polymer collapse, which can be modulated by pH, concentration, ELP guest residue, and ELP chain length. ELPs can be coupled with antiparallel, complementary alpha-helical proteins, like the CCE and CCK peptides which can form coiled-coils. ELP fusion proteins of varying length and biorecognizable peptides (CCE or CCK) were genetically engineered, produced recombinantly using E. coli, purified via inverse transition cycling (ITC), and characterized. These proteins were confirmed via amino acid analysis, matrix assisted laser desorption/ionization (MALDI-TOF), sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). Confirmed proteins were analyzed using dynamic light scattering, circular dichroism, turbidimetry, and precipitation assays. Results demonstrated an inverse correlation between chain length, concentration, and ionic strength with LCST, canonical of ELP properties. Additionally, CCE/CCK fusion protein mixtures generated varying responses, compared to single component solutions. CCE-ELPs can remain in a more soluble state than ELPs alone, in an LCST condition. This study has shown the capability to produce several ELP fusion proteins recombinantly. Furthermore, the study documents protein collapse during temperature increase, CCE solubility tag performance, and LCST under ELP chain length or concentration increase. The thermoresponsive nature demonstrated by these fusion proteins shows their potential as DFMTs and future studies will evaluate clustering at the cell surface.

Additional Abstract Information

Presenter: Blair Armstrong

Institution: University of Utah

Type: Poster

Subject: Biological & Chemical Engineering

Status: Approved

Time and Location

Session: Poster 2
Date/Time: Mon 3:00pm-4:00pm
Session Number: 2556