(T0930-01-04) Zinc-Associated Peptide Coacervates for Recombinant Protein Capture and Release

Purpose: This research aimed to develop a delivery platform for loading and extended release of recombinant proteins. [1, 2]. To this end, the self-assembling peptide (SAP) “EAKH6” was used to form coacervates of β-fibrils [3], which in the presence of zinc ions (Zn²⁺), would capture proteins co-expressed with his-tags. The premise is that the hexa-histidine residues in EAKH6 form coordination bonds with Zn²⁺, which further enhances the fibrillization of the SAP. The resulting Zn-bound coacervates also provide a mechanism for loading his tagged proteins [4, 5]. In this study we used a His-tagged Green Fluorescent Protein (hisGFP) to probe an EAKH6: Zn coacervate system in order to understand its loading and release properties.

Methods: The study explores the formation of Zn²⁺ with EAKH6 coacervates at molar ratio of 10:1 and 1:1, and the role of coordination bond formation in protein loading. HisGFP was incorporated to investigate the protein loading in coacervates with increased florescence intensities. His-Zn2+ interactions for coordination bond was tested by disrupting with EDTA, and replacing EAKH6 with EAK16. Addition of excess imidazole (240 mM) to displace the His-Zn2+ interactions further demonstrates the His interaction with Zn²⁺ in coordination bond formation. Protein release was measured in both Zn²⁺:EAKH6 and Ca²⁺:EAKH6 coacervates. Presence of Zn²⁺ was verified by staining with TSQ and florescence microscopy.

Results: Compared to the 1:1 molar ratio of Zn²⁺ to EAKH6, the 10:1 formulated exhibited increased loading and extended release of hisGFP. The rationale for using an excess Zn was to ensure constant interactions among EAKH6:Zn: hisGFP (Figure 1a -1b) [5]. Loading of hisGFP was significantly reduced in the absence of Zn or in the presence of EDTA, a divalent metal chelator. Loading was also reduced in coacervates formed with a EAK SAP without histidine residues. In the presence of imidazole, the amount of hisGFP associated with Zn²⁺: EAKH6 coacervates was reduced. The capture of hisGFP was reversible, as 27% of the loaded protein was released within 5 days and up to 90% in 45 days. Direct evidence of bound Zn in the coacervates was demonstrated using fluorescent microscopic imaging of samples rinsed extensively (Figure 1e & f).

Conclusion: These results support the existence of Zn²⁺: EAKH6 coacervates and zinc-mediated loading and release of His-tagged proteins.

References: 1. Koutsopoulos, S., et al., Controlled release of functional proteins through designer self-assembling peptide nanofiber hydrogel scaffold. Proceedings of the National Academy of Sciences, 2009. 106(12): p. 4623-4628.
2. Wu, J., et al., Challenges in delivering therapeutic peptides and proteins: A silk-based solution. Journal of Controlled Release, 2022. 345: p. 176-189.
3. Gelain, F., Z. Luo, and S. Zhang, Self-assembling peptide EAK16 and RADA16 nanofiber scaffold hydrogel. Chemical reviews, 2020. 120(24): p. 13434-13460.
4. Promsuk, J., et al., Optimizing protein delivery rate from silk fibroin hydrogel using silk fibroin-mimetic peptides conjugation. Scientific Reports, 2024. 14(1): p. 4428.
5. Barraud, P., M. Schubert, and F.H.-T. Allain, A strong 13 C chemical shift signature provides the coordination mode of histidines in zinc-binding proteins. Journal of biomolecular NMR, 2012. 53: p. 93-101.
6. Liu, W., et al., Local retention of antibodies in vivo with an injectable film embedded with a fluorogen-activating protein. Journal of controlled release, 2016. 230: p. 1-12.


His tagged GFP protein loading in Zn2+: EAKH6 coacervate. a) 10:1 molar ratio of Zn2+ with His (EAKH6) displays increased protein loading. b) 1:1 molar ratio of Zn2+ with His (EAKH6) with decreased protein loading.


c, d) shows the In-vitro protein release from Zn2+: EAKH6 and it is compared with control group EAKH6 weak electrostatic interactions with calcium. Protein release depends on binding affinity between Zn2+ and His, which is reversible


e) represents florescence microscopic evaluation of Zn2+: EAKH6, compared to f) Zn2+ interaction EAKH16.