Amine Landscaping to Maximize Protein-Dye Fluorescence and Ultrastable Protein-Ligand Interaction

Jacobsen, Michael T.; Fairhead, M.; Fogelstrand, Per; Howarth, Mark

doi:10.1016/j.chembiol.2017.06.015

Cited by 14 publications

(17 citation statements)

References 60 publications

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“…Using this approach, a binding affinity of K d = 7.6 ± 1.2 pM was obtained. This K d value is roughly two orders of magnitude larger than that measured for wild-type streptavidin-biotin ( K d = 10 −14 M) 32 and comparable ( K d = 10 −11 M) 33 to cases where the association may be affected by dye conjugation 34 . Attachment of biotin moieties to a larger group can also restrict its free diffusion and thus reduce the binding affinity 35 .…”

Section: Resultsmentioning

confidence: 58%

Small molecule electro-optical binding assay using nanopores

Sze²,

et al. 2019

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The identification of short nucleic acids and proteins at the single molecule level is a major driving force for the development of novel detection strategies. Nanopore sensing has been gaining in prominence due to its label-free operation and single molecule sensitivity. However, it remains challenging to detect small molecules selectively. Here we propose to combine the electrical sensing modality of a nanopore with fluorescence-based detection. Selectivity is achieved by grafting either molecular beacons, complementary DNA, or proteins to a DNA molecular carrier. We show that the fraction of synchronised events between the electrical and optical channels, can be used to perform single molecule binding assays without the need to directly label the analyte. Such a strategy can be used to detect targets in complex biological fluids such as human serum and urine. Future optimisation of this technology may enable novel assays for quantitative protein detection as well as gene mutation analysis with applications in next-generation clinical sample analysis.

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Section: Resultsmentioning

confidence: 58%

Small molecule electro-optical binding assay using nanopores

Sze²,

et al. 2019

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“… 18 23 mg/L is close to the maximum we achieve for any soluble protein in shake-flask E. coli culture. 20 , 42 , 43 Higher yields should be accessible through fermentation. 44 …”

Section: Results and Discussionmentioning

confidence: 99%

Engineering a Rugged Nanoscaffold To Enhance Plug-and-Display Vaccination

et al. 2018

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Nanoscale organization is crucial to stimulating an immune response. Using self-assembling proteins as multimerization platforms provides a safe and immunogenic system to vaccinate against otherwise weakly immunogenic antigens. Such multimerization platforms are generally based on icosahedral viruses and have led to vaccines given to millions of people. It is unclear whether synthetic protein nanoassemblies would show similar potency. Here we take the computationally designed porous dodecahedral i301 60-mer and rationally engineer this particle, giving a mutated i301 (mi3) with improved particle uniformity and stability. To simplify the conjugation of this nanoparticle, we employ a SpyCatcher fusion of mi3, such that an antigen of interest linked to the SpyTag peptide can spontaneously couple through isopeptide bond formation (Plug-and-Display). SpyCatcher-mi3 expressed solubly to high yields in Escherichia coli, giving more than 10-fold greater yield than a comparable phage-derived icosahedral nanoparticle, SpyCatcher-AP205. SpyCatcher-mi3 nanoparticles showed high stability to temperature, freeze–thaw, lyophilization, and storage over time. We demonstrate approximately 95% efficiency coupling to different transmission-blocking and blood-stage malaria antigens. Plasmodium falciparum CyRPA was conjugated to SpyCatcher-mi3 nanoparticles and elicited a high avidity antibody response, comparable to phage-derived virus-like particles despite their higher valency and RNA cargo. The simple production, precise derivatization, and exceptional ruggedness of this nanoscaffold should facilitate broad application for nanobiotechnology and vaccine development.

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“…To make use of this convenient set of reagents, we next developed a simple protocol to convert NHS-esters to N-terminal proline specific reagents using tyramine. Biotin was chosen as an initial target for methodology development because it is commonly used to tag proteins for fluorescence labeling, 47 affinity capture, 48 and surface immobilization. 49 To prepare biotin-phenol 1a, a 1.1 equivalent portion of the corresponding NHS-ester was added to tyramine in dry DMF, Figure 3a.…”

Section: Resultsmentioning

confidence: 99%

Enzymatic Modification of N-Terminal Proline Residues Using Phenol Derivatives

et al. 2019

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A convenient enzymatic strategyis reported for the modification of proline residues in the N-terminal positions of proteins. Using a tyrosinase enzyme isolated from Agaricus bisporus(abTYR), phenols and catechols are oxidized to highly reactive o-quinone intermediates that then couple to N-terminal proline residues in high yield. Key advantages of this bioconjugation method include (1) the use of air-stable precursors that can be prepared on large scale if needed, (2) mild reaction conditions, including low temperatures, (3) the targeting of native functional groups that can be introduced readily on most proteins, and (4) the use of molecular oxygen as the sole oxidant. This coupling strategy was successfully demonstrated for the attachment of a variety of phenol-derivatized cargo molecules to a series of protein substrates, including self-assembled viral capsids, enzymes, and a chitin binding domain (CBD). The ability of the CBD to bind to the surfaces of yeast cells was found to be unperturbed by this modification reaction. File list (2) download file view on ChemRxiv Maza 2018. ChemRxiv Final.pdf (0.97 MiB) download file view on ChemRxiv SI. Maza. ChemRxiv.pdf (1.50 MiB)

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Amine Landscaping to Maximize Protein-Dye Fluorescence and Ultrastable Protein-Ligand Interaction

Cited by 14 publications

References 60 publications

Small molecule electro-optical binding assay using nanopores

Small molecule electro-optical binding assay using nanopores

Engineering a Rugged Nanoscaffold To Enhance Plug-and-Display Vaccination

Enzymatic Modification of N-Terminal Proline Residues Using Phenol Derivatives

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