Development of Mixed-Charge EK Polypeptides for Protein Protection

Abstract

The presented work focuses on the development of mixed charge polypeptides and their fusion proteins and protein conjugates for the protection of proteins. Compared to many polymers used to modify proteins, polypeptides are an ideal candidate for protein modification due to their biocompatibility, biodegradability, and uniformity. Polypeptides composed of alternating glutamic acid and lysine residues (EK) function as rationally-designed polypeptide analogues of low-fouling zwitterionic polymers due to their ability to resist nonspecific protein adsorption. Zwitterionic polymers have demonstrated superior properties related to protein protection, pharmacokinetics, and immunogenicity compared to other polymers, including poly(ethylene glycol) (PEG), which has been widely used to modify therapeutic proteins in the clinic. As EK has demonstrated similar zwitterionic properties it can be used in similar capacities when used to modify therapeutic proteins. The use of EK allows for the use of recombinant protein expression techniques, thereby allowing for the synthesis of well-defined products of uniform composition without any inherent batch to batch variation. Additionally, recombinant techniques allow for EK to be a component of a fusion protein to modify target therapeutic proteins, allowing for one-step generation of any number of EK-modified products. This work develops the capabilities of EK through various systems from proof of concept through in vivo capabilities and demonstrates that EK behaves similarly to zwitterionic polymers when used to modify proteins. A proof of concept system utilized a model β-lactamase system to demonstrate the generation of fusion proteins containing EK. These fusion proteins were able to be expressed at a uniform molecular weight and purified. It was observed that EK demonstrated the conferring of the same protecting properties in vitro against environmental stressors and improvements to enzyme kinetics that zwitterionic polymers had previously demonstrated. These in vitro properties of EK were again demonstrated when EK was used as a part of a fusion protein with organophosphate hydrolase. Additionally EK exhibited its zwitterionic low-fouling characteristics by preventing the formation of the dimeric structure of wild-type organophosphate hydrolase and leaving stable monomeric structures, demonstrating the ability of EK to protect the surface of the protein from foreign interactions. This effort also represents the first known expression of a stable monomeric unit of the protein. Furthermore, other EK fusion protein systems and standalone EK peptides have also been expressed and studied.