Therapeutic proteins frequently need to be modified with high-molecular-weight molecules to improve their pharmacokinetic properties. The genetic linkage of therapeutic proteins to a high-molecular-weight zwitterionic peptide, termed EKP, offers a promising approach. As with any protein modification, EKP could impact the structural behavior and receptor binding properties of the linked therapeutic protein, thereby altering its bioactivity. To evaluate the effects of EKP on therapeutic proteins, we study the receptor binding properties of high-molecular-weight EKP linked to the growth colony-stimulating factor (GCSF) using the genetically based yeast display platform. We find that yeast-displayed EKP-GCSF and GCSF exhibits similar binding to its receptor GCSF-R, suggesting that EKP does not hinder receptor binding. Furthermore, yeast-displayed EKP-GCSF demonstrates protection against thermal denaturation compared to GCSF. Similarly, to study the structural effects of EKP on GCSF, we employ in silico modeling using alphaFold2 in conjunction with molecular dynamics (MD) simulations. Likewise, in silico modeling reveals that EKP does not alter the structural behavior of GCSF. Finally, we demonstrate the functional benefits of EKP, by which the EKP-GCSF fusion protein produced in Escherichia coli exhibits improved pharmacokinetics and prolonged bioactivity in vivo.
|Number of pages||9|
|State||Published - Aug 17 2022|
Bibliographical noteFunding Information:
S.J. acknowledges start-up support from Cornell University, including Robert S. Langer Professorship and Cornell NEXT Nano Initiative, and the National Science Foundation (DMR1708436). Q.Q and Q.S acknowledge the support of Start-up funds provided by the University of Kentucky. The simulations were conducted on the computational facilities provided by the High-Performance Computing Center of the University of Kentucky.
© 2022 American Chemical Society.
ASJC Scopus subject areas
- Biomedical Engineering
- Pharmaceutical Science
- Organic Chemistry