(781e) Using Poly(EK) Fusion Proteins to Enhance Protein Stability

Liu, E. J., University of Washington
Sinclair, A., University of Washington
Jiang, S., University of Washington
Protein PEGylation has been used for several decades, and has resulted in the development of numerous PEGylated protein drugs. PEGylation typically improves protein stability at the expense of bioactivity, biodegradability, or immunogenicity by forming a shielding layer around the protein. Recently, ultra low fouling zwitterionic polymers, including poly(carboxybetaine) (pCB), have demonstrated the ability to improve the thermal and chemical stabilities of proteins when chemically conjugated to protein surfaces. Oppositely charged glutamic acid (E) and lysine (K) and residues have been shown to occur on protein surfaces at balanced ratios, and alternating repeating poly(EK) sequences have demonstrated nonfouling zwitterionic characteristics when utilized in functionalizing surfaces and nanoparticles. Thus EK ideally presents itself as a naturally-derived pCB analogue, allowing for the use of recombinant techniques for one-step production of protein-poly(EK) fusion proteins through protein expression techniques.

Genes encoding for poly(EK) tails of 10 kD and 30 kD have been appended to the C-terminus of proteins. The resulting fusion protein products containing the poly(EK) tails were expressed in E. coli strain BL21(de3) and purified. Enzyme kinetics and activity assays and thermal and salt stability assays were performed to demonstrate the effect that poly(EK) had on improving protein stability. The addition of poly(EK) to native β-lactamase and a destabilized TEM-19 mutant indicated that the poly(EK) tail was not significantly detrimental to protein activity, with fusion protein products containing the 10 kD poly(EK) maintaining native activity. Additionally the addition of poly(EK) increased the fusion protein productâ??s affinity to the substrate, with appended 10 kD and 30 kD poly(EK) reducing the Km from 70 to 25 and 45 μM, respectively. Similarly to pCB, the addition of poly(EK) was able to improve the proteinâ?? thermal stability. The addition of 10 kD and 30 kD poly(EK) increased the midpoint transition temperature by approximately 15 and 20 °C, respectively. Interestingly, the addition 10 kD poly(EK) did not change the profile of the thermal stability curve while increasing stability, whereas the addition of 30 kD poly(EK) generated a modified stability profile. Additionally, proteins containing 30 kD (polyEK) were able to retain more than 30% of initial activity after 1 hour of incubation at 95°C, a nearly 300% improvement over the unmodified wild-type protein. This system can be applied to any protein where increased stability is desired, and is being used to study more application-relevant systems. Additionally further study will allow for probing the effect of poly(EK) on protein structure, allowing for the study of its effects on the protein product's tertiary and quarternary structure and poly(EK)'s secondary structure. Recently it has been demonstrated that PEGylated drugs are beginning to see accelerated clearance, which may be tied to the increased presence of anti-PEG antibodies. This allows for a clear demonstration of the effect of poly(EK) in vivo to study protein circulation and immunogenicity.


[1] Liu, E. J.; Sinclair, A.; Keefe, A. J.; Nannenga, B., L.; Coyle, B. L., Baneyx, F, Jiang, S., Biomacromolecules, 2015
[2] Keefe, A. J.; Jiang, S., Nature Chemistry, 2012, 4, 59-63.