(C-101) Single-Step N-Functionalization and Purification of Bioactive Proteins for Biomaterial Modification

Introduction:: Creating protein-functionalized biomaterials has historically been hampered by harsh modification and conjugation chemistries, leading to severely reduced construct bioactivity and significant batch-to-batch variability. To that end, creating well-defined polypeptide conjugates and assemblies is critical to manifold applications requiring highly nuanced control over spatiotemporal epitope presentation. Inspired by recent advances in chemoenzymatic semisynthesis modalities, we demonstrate a method to purify and modify recombinant proteins with synthetic handles necessary to site-specifically tether them into biomaterials via many common chemistries (e.g., thiol-ene, SPAAC, oxime, Michael-type) in a single step. To establish its broad applicability, we demonstrate the use of this platform for the modification of fluorescent proteins, model enzymes, and growth factors, all while retaining native-level bioactivity. We predict that this method will enable a fast and tractable strategy for the creation of a suite of site-specifically functionalized proteins, with direct implications in biomaterials science and tissue engineering.


Materials and Methods:: Protein constructs were cloned into plasmid expression vectors using established molecular cloning approaches and expressed through general bacterial fermentation techniques, allowing for user control over scale and ultimate yield. Requisite peptide sequences endowed with canonical and/or non-canonical residues were all synthesized through microwave-assisted solid-phase peptide synthesis, purified and aliquoted for downstream use. Reaction conditions for functionalization / purification were monitored over time using SDS-PAGE gel analysis and reaction progress was monitored and quantified through gel densitometry.


Results, Conclusions, and Discussions:: Our proposed method represents a “plug-and-play” approach for the generation of pristine site-specifically modified proteins with negligible impact on protein bioactivity. Reaction properties such as kinetics, temperature-dependence, and reactant stoichiometry were all analyzed and characterized through gel densitometry analyses, demonstrating the robustness of our platform across a range of possible conditions. Functionalization yields are near-quantitative within two hours, and robustness with respect to temperature conditions renders it a particularly suitable method for the modification of highly thermo-sensitive proteins. Finally, we show that our platform is compatible with a variety of targets such as fluorescent markers, model enzymes, and growth factors, and demonstrate that bioactivity is preserved for all proteins-of-interest through assays that are tuned to each. All final constructs are purified to very high purities ( >99%) as evinced by SDS-PAGE.

We envision that this platform will find broad applicability within the biomaterials community for routine generation of site-specifically modified biomacromolecules in a nearly-scarless manner. Our platform has proven successful for the functionalization of a variety of potential targets, all while side-stepping the use of aphysiological additives, denaturing agents, or otherwise harsh reaction conditions. Work currently underway within our lab focuses on identifying biologically challenging targets that have so far proven refractory to site-specific modification and applying them as targets to this platform, with an eye towards addressing their function within the context of cell-laden hydrogel biomaterials.


Acknowledgements (Optional): : RTG would like to acknowledge funding support from the John C. Berg Endowed Fellowship. Additional funding for this work was provided by the NIH (R35GM138036) and NSF (DMR 1652141).


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