Macromolecular assembly of bioluminescent protein nanoparticles for enhanced imaging

Li, Enya; Brennan, Caroline K.; Ramírez, A. Gonzaga; Tucker, Jo A.; Butkovich, Nina; Meli, Vijaykumar S.; Ionkina, Anastasia A.; Nelson, Edward L.; Prescher, Jennifer A.; Wang, Szu-Wen

doi:10.1016/j.mtbio.2022.100455

Cited by 2 publications

(2 citation statements)

References 78 publications

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“…Another area of research where NLuc has been utilized extensively is in the development of protein fragment complementation assay, typically utilized for monitoring protein-protein interaction in living cells. 24-26 Additional applications of NLuc include nanoparticle conjugates utilized for in vivo imaging 27 , sensitive viral infection detection 28 , to evaluate one of metabolic intermediate in fatty acid metabolism 29 or to test antibiotic susceptibility 30 . Further, NLuc has been successfully applied in the development of various Bioluminescence Resonance Energy Transfer (BRET)-based biomolecular assays 4-7, 31-33 such as to study molecular/organelle interactions 34, 35 , for optogenetics purposes 36 , for drug descovery 37, 38 , as genetic encoded light source for a photodynamic anticancer therapy 39 , biomolecule detection 40 , monitoring molecular tension 41 , and assays to monitor SARS-CoV-2 protease activity 42, 43 .…”

Section: Introductionmentioning

confidence: 99%

A Slow but Steady NanoLuc: R162A mutation results in a decreased, but stable, NanoLuc activity

Ahmed

Geethakumari

Sultana

et al. 2023

Preprint

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NanoLuc (NLuc) luciferase has found extensive application in designing a range of biological assays including gene expression analysis, protein-protein interaction and protein conformational changes due to its enhanced brightness and small size. However, questions related to its mechanism of interaction with the substrate, furimazine, as well as bioluminescence activity remains elusive. Here, we combined molecular dynamics (MD) simulation and mutational analysis to show that the R162A mutation results in a decreased but stable bioluminescence activity of NLuc in vitro. Specifically, we performed multiple, all-atom, explicit solvent MD simulations of the apo and furimazine-docked (holo) NLuc structures revealing differential dynamics of the protein in the absence and presence of the ligand. Further, analysis of trajectories for hydrogen bonds (H-bonds) formed between NLuc and furimazine revealed substantial H-bond interaction between R162 and Q32 residues. Mutation of the two residues in NLuc revealed a decreased but stable activity of the R162A, but not Q32A, mutant NLuc in in vitro assays performed with furimazine. In addition to highlighting the role of the R162 residue in NLuc activity, we believe that the mutant NLuc will find wide application in designing in vitro assays requiring extended monitoring of NLuc bioluminescence activity.

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Section: Introductionmentioning

confidence: 99%

A Slow but Steady NanoLuc: R162A mutation results in a decreased, but stable, NanoLuc activity

Ahmed

Geethakumari

Sultana

et al. 2023

Preprint

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“…Direct genetic fusion of protein antigens onto virus-like particles has shown some success with particular platforms and therefore was explored with the E2 protein nanoparticle; however, expression and correct folding into a soluble protein assembly needs to be empirically tested. ,− The introduction of polyhistidine tags on recombinant proteins to bind to Ni-NTA-based matrices is a well-established protein purification methodology, − and we previously applied this complexation-based approach in nanoparticle-mediated delivery of influenza hemagglutinin antigen; here, we used it as the basis for loading polyhistidine-tagged (His-tag) CBU1910 antigen onto E2 NPs. To attach protein antigens, covalently and modularly, onto the surface of E2 NPs, the versatile protein–protein conjugation method, SpyTag/SpyCatcher, was implemented. − The adaptive immune response (i.e., antibody and T cell responses) to the most favorable NP construct was then examined to determine the prophylactic potential of the vaccine formulation.…”

Section: Introductionmentioning

confidence: 99%

Engineering Protein Nanoparticles Functionalized with an Immunodominant Coxiella burnetii Antigen to Generate a Q Fever Vaccine

Ramirez,

Felgner,

Jain

et al. 2023

Bioconjugate Chem.

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Coxiella burnetii is the causative agent of Q fever, for which there is yet to be an FDA-approved vaccine. This bacterial pathogen has both extra- and intracellular stages in its life cycle, and therefore both a cell-mediated (i.e., T lymphocyte) and humoral (i.e., antibody) immune response are necessary for effective eradication of this pathogen. However, most proposed vaccines elicit strong responses to only one mechanism of adaptive immunity, and some can either cause reactogenicity or lack sufficient immunogenicity. In this work, we aim to apply a nanoparticle-based platform toward producing both antibody and T cell immune responses against C. burnetii. We investigated three approaches for conjugation of the immunodominant outer membrane protein antigen (CBU1910) to the E2 nanoparticle to obtain a consistent antigen orientation: direct genetic fusion, high affinity tris-NTA-Ni conjugation to polyhistidine-tagged CBU1910, and the SpyTag/SpyCatcher (ST/SC) system. Overall, we found that the ST/SC approach yielded nanoparticles loaded with the highest number of antigens while maintaining stability, enabling formulations that could simultaneously co-deliver the protein antigen (CBU1910) and adjuvant (CpG1826) on one nanoparticle (CBU1910-CpG-E2). Using protein microarray analyses, we found that after immunization, antigen-bound nanoparticle formulations elicited significantly higher antigen-specific IgG responses than soluble CBU1910 alone and produced more balanced IgG1/IgG2c ratios. Although T cell recall assays from these protein antigen formulations did not show significant increases in antigen-specific IFN-γ production compared to soluble CBU1910 alone, nanoparticles conjugated with a CD4 peptide epitope from CBU1910 generated elevated T cell responses in mice to both the CBU1910 peptide epitope and whole CBU1910 protein. These investigations highlight the feasibility of conjugating antigens to nanoparticles for tuning and improving both humoral- and cell-mediated adaptive immunity against C. burnetii.

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Macromolecular assembly of bioluminescent protein nanoparticles for enhanced imaging

Cited by 2 publications

References 78 publications

A Slow but Steady NanoLuc: R162A mutation results in a decreased, but stable, NanoLuc activity

A Slow but Steady NanoLuc: R162A mutation results in a decreased, but stable, NanoLuc activity

Engineering Protein Nanoparticles Functionalized with an Immunodominant Coxiella burnetii Antigen to Generate a Q Fever Vaccine

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