Poster O12 - Temperature-triggered assembly of protein hydrogels via an N-glycosylation consensus sequence polypeptide domain

Introduction: Certain amino acid sequences constitute polypeptides that form supramolecular networks in response to a specific stimulus, such as temperature. These sequences can then be genetically fused to protein cargo and assembled after recombinant expression to create functional biomaterials. Here, we have generated an N-glycosylation tag, (GGGSGGGSGGNWTT)10 or “NGT,” fused to either Superfolder green fluorescent protein (NGTsfGFP) or NanoLuc luciferase (NGTnL) that assembles into a supramolecular network when cooled after recombinant expression. NGT is a repeat of a consensus sequence for Actinobacillus pleuropneumoniae N-glycosyltransferase (ApNGT). At high concentration, NGT fusion proteins generated bioactive viscoelastic gels that were disrupted by the chaotropic agent sodium thiocyanate, asparagine-to-glutamine mutation of the NGT sequence (“QGT”), or enzymatic glycosylation of the asparagine residues. These studies suggested that the repetitive asparagine residues in the NGT sequence provide intermolecular non-covalent interactions that contribute to the temperature-dependent assembly of the fusion protein. Co-assembly of NGTsfGFP and NGTnL was demonstrated by bioluminescence resonance energy transfer (BRET) between the fused protein domains.

Materials and

Methods: NGTsfGFP, NGTnL, ApNGT, and QGTsfGFP were generated using recombinant bacterial expression. Briefly, pET-21d(+) plasmids encoding the target proteins were transformed into Origami™ B(DE3) competent cells, expressed via isopropyl β-D-1-thiogalactopyranoside induction, and purified via immobilized metal affinity chromatography. NGTsfGFP and NGTnL gels were formed by freezing purified protein at -80 °C, thawing at 4 °C, and centrifuging the resulting assembled protein. For rheological measurements, 80 μL of gel was extruded into gel molds and loaded onto an Anton Paar MCR302 with 8 mm diameter parallel plates. Frequency sweeps were performed at 4 °C, 37 °C, and 55 °C to determine the storage modulus (G’) and loss modulus (G”). Step-strain and step-shear flow measurements were performed on 1 mM NGTsfGFP and 3 mM NGTnL at 37 °C. Viscosity was measured at alternating shear rates of 0.5 Hz and 100 Hz, and storage and loss moduli were recorded at alternating 0.3% strain and 1000% strain. Turbidity measurements were performed at an absorbance wavelength of 600 nm from 4 °C to 55 °C. NGTsfGFP was glycosylated using 25 molar excess of UDP-Glucose in the presence of ApNGT and confirmed via MALDI-TOF mass spectrometry. BRET assay measurements on co-assembled NGTsfGFP and NGTnL were made by measuring luminescence and fluorescence emissions from 400-700 nm in the presence of the NanoLuc luciferase substrate furimazine.

Results, Conclusions, and Discussions: NGTsfGFP and NGTnL formed weak viscoelastic gels at 1 mM and 3 mM that were weak viscoelastic gels at all measured frequencies and temperatures, as determined by the ratio of G” to G’. G’ and G” decreased with increasing temperature, indicating that heat softened the gels. Glycosylation of NGTsfGFP (Glc-NGTsfGFP) was confirmed by the presence of a mass shift of the expected molecular weight measured by MALDI-TOF mass spectrometry. Solutions of 500 µM Glc-NGTsfGFP were completely soluble from 4 °C to 55 °C, whereas 500 µM was a gel under these same conditions. Similarly, QGTsfGFP failed to form a gel at 500 µM and, and turbidity measurements showed little to no assembly of QGTsfGFP when compared to NGTsfGFP at the same conditions. In the presence of sodium thiocyanate, NGTsfGFP rapidly disassembled when heated, which was observed through multiple cycles of heating and cooling. NGTsfGFP and NGTnL partially recovered from alternating high shear, and rapidly reformed into a gel after extrusion through a 25-gauge syringe needle. After application of 1000% strain, NGTsfGFP recovered 81% of its initial stiffness after 10 minutes, and NGTnL recovered 94% of its initial stiffness in the same amount of time. Upon addition of furimazine to an NGTsfGFP and NGTnL co-assembled gel, emission of blue light from NGTnL and green light from NGTsfGFP was observed. This suggests that the protein pairs are within 100 Å of one another. These data support that NGT is a polypeptide tag that can mediate the assembly of fused proteins into bioactive supramolecular gels. Due to the injectability and self-healing of these gels, they may find application as minimally-invasive delivery vehicles. Future work will involve further characterization of the molecular drivers of NGT phase separation, and generating NGT-based biomaterials with different protein fusion partners.

Acknowledgements (Optional): This research was supported by the National Institute of Health. Support to B.G.K is acknowledged from NIDDK 5 R01 DK129690-03 and NIAID 5 R01 AI171045-02. Support to G.A.H is acknowledged from NIGMS 1 R35 GM133697-01. We would like to acknowledge the University of Florida Mass Spectrometry Research and Education Center for technical assistance and training with MALDI-TOF MS, which is funded by NIH S10 OD021758-01A1 and S10 OD030250-01A1.