2024 AIChE Annual Meeting
Investigating the Role of Net Charge and Charge Patterning in the Encapsulation of Green Fluorescent Protein in Complex Coacervates
Complex coacervation is the liquid-liquid phase separation of oppositely charged polyelectrolyte solutions resulting in a polymer-rich coacervate phase and a polymer-poor supernatant phase. Complex coacervation has numerous applications, ranging from the formation of membraneless organelles to drug delivery. Coacervates can also be formed with protein solutions in which the polyelectrolytes interact with the protein and incorporate it into the polymer-rich phase. Protein encapsulation is especially interesting in fields like food science and drug delivery, where the sequestration of proteins allows for a much greater concentration than in solution while still maintaining solubility and protein viability. Prior studies on protein encapsulation have found that the net charge of proteins plays a major role in how they interact with polyelectrolytes and how effectively they are sequestered into the coacervates. Existing research also shows that the charge patterning of surface amino acid residues affects protein encapsulation along with pH and salt concentration. We seek to explore the convolution of these factors on protein incorporation through the use of tailored supercharged green fluorescent proteins (GFPs). Supercharged GFPs can be engineered to have a specific net charge by altering specific amino acid residues, and this tailoring can be used to control the patterning of charge on the protein surface. In this study, we will explore the incorporation of GFP variants with different net charges and charge distributions into complex coacervates made with poly-L-lysine and poly-D,L-glutamate. The incorporation will be assessed by measuring the turbidity and fluorescence of coacervates and calculating the concentration of protein in the coacervate versus the supernatant. Lastly, thermal stability assays will be performed to assess the temperature at which the GFPs become denatured. These results will give insight into how different parameters of GFPs affect protein encapsulation. This insight can be used as a foundation in future applications of GFPs, but can also be extended to encapsulation studies of other biologics ranging from other proteins to viruses to bacteria.