Conjugation of DNA with protein using His-tag chemistry and its application to the aptamer-based detection system

Shimada, Josui; Maruyama, Tatsuo; Hosogi, Takuya; Tominaga, Jo; Kamiya, Noriho; Goto, Masahiro

doi:10.1007/s10529-008-9784-4

Cited by 30 publications

(18 citation statements)

References 20 publications

(19 reference statements)

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“…25 ODFs chemically resemble DNA, and thus one might make use of methodologies of protein conjugation that have been developed for DNA itself; 26-29 however, to our knowledge, DNA has yet to be conjugated to proteins via the haloalkane dehalogenase approach. Here we have developed a general strategy for genetically-encoded labeling of proteins with ODF fluorophores (and potentially with DNA as well), by employing the HaloTag haloalkane dehalogenase enzyme.…”

Section: Introductionmentioning

confidence: 99%

Genetically Encoded Multispectral Labeling of Proteins with Polyfluorophores on a DNA Backbone

2013

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Genetically-encoded methods for protein conjugation are of high importance as biological tools. Here we describe the development of a new class of dyes for genetically encoded tagging that add new capabilities for protein reporting and detection via HaloTag methodology. Oligodeoxyfluoroside (ODFs) are short DNA-like oligomers in which the natural nucleic acid bases are replaced by interacting fluorescent chromophores, yielding a broad range of emission colors using a single excitation wavelength. We describe the development of an alkyl halide dehalogenase-compatible chloroalkane linker phosphoramidite derivative that enables the rapid automated synthesis of many possible dyes for protein conjugation. Experiments in vitro test enzymatic self-conjugation of nine different DNA-like dyes to proteins with HaloTag domains, and the data confirm rapid and efficient covalent labeling of proteins. Notably, a number of the ODF dyes are found to increase in brightness or change color upon protein conjugation. Tests in mammalian cellular settings reveal that the dyes are functional in multiple cellular contexts, both on the cell surface and within the cytoplasm, allowing protein localization to be imaged in live cells by epifluorescence and laser confocal microscopy.

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Section: Introductionmentioning

confidence: 99%

Genetically Encoded Multispectral Labeling of Proteins with Polyfluorophores on a DNA Backbone

2013

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“…Ni 2+ -NTA has been used to immobilize His 6 -tagged proteins on agarose beads [2], microtiter plates [4], and lipid surfaces [5 - 9]. It has also been exploited in protein-labeling schemes where Ni 2+ -NTA has been coupled to horseradish peroxidase [10], oligonucleotides [11; 12], biotin [13], and nano-gold particles [14; 15]. This plethora of applications of the Ni 2+ -NTA/His 6 -tag technology is a testament to the value and flexibility of this metal chelation system.…”

Section: Introductionmentioning

confidence: 99%

Hexahistidine-tag-specific optical probes for analyses of proteins and their interactions

Zhao

Hellman

Zhan

et al. 2010

Analytical Biochemistry

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The hexahistidine (His6)/Nickel (II)-Nitrilotriacetic Acid (Ni2+-NTA) system is widely used for affinity purification of recombinant proteins. The NTA group has many other applications, including the attachment of chromophores, fluorophores, or nano-gold to His6-proteins. Here we explore several applications of the NTA-derivative, (Ni2+-NTA)2-Cy3. This molecule binds our two model His6-proteins, N-ethylmaleimide Sensitive Factor (NSF) and O6-alklyguanine-DNA alkyltransferase (AGT), with moderate affinity (K ∼ 1.5 × 106 M-1) and no effect on their activity. Its high specificity makes (Ni2+-NTA)2-Cy3 ideal for detecting His6-proteins in complex mixtures of other proteins, allowing (Ni2+-NTA)2-Cy3 to be used as a probe in crude cell extracts and as a His6-specific gel stain. (Ni2+-NTA)2-Cy3 binding is reversible in 10 mM EDTA or 500 mM imidazole but in their absence, it exchanges slowly (kexchange ∼ 5 × 10-6 s-1 with 0.2 μM labeled protein in the presence of 1μM His6-peptide). Labeling with (Ni2+-NTA)2-Cy3 allows characterization of hydrodynamic properties by fluorescence anisotropy or analytical ultracentrifugation under conditions (e.g. high ADP absorbance) that prevent direct detection of protein. In addition, fluorescence resonance energy transfer (FRET) between (Ni2+-NTA)2-Cy3-labeled proteins and suitable donors/acceptors provides a convenient assay for binding interactions and for measurements of donor-acceptor distances.

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“…On the other end of the tether, the protein of interest is either covalently linked through a thiol-maleimide crosslink (23) or tethered non-covalently via the histidine tag to Anti-his antibodies, which in turn are covalently linked to aminated DNA tethers (29). The histidine tag of proteins was used previously to non-covalently link to DNA (35–38) and directly attaching proteins to NTA-modified AFM tips (39). It was demonstrated that the His-Ni-NTA bond is stable enough (~120pN at 400pN/s) to facilitate single-molecule force spectroscopy (SMFS) experiments of the tethered protein (40–43) thus allowing AFM-based studies measuring unbinding forces of CBM3a using Ni-NTA (25),(27).…”

Section: Introductionmentioning

confidence: 99%

Acoustic Force Spectroscopy Reveals Subtle Differences in Cellulose Unbinding Behavior of Carbohydrate-Binding Modules

Hackl

Contrada

Ash

et al. 2021

Preprint

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To rationally engineer more efficient cellulolytic enzymes for cellulosic biomass deconstruction into sugars for biofuels production, it is necessary to better understand the complex enzyme-substrate interfacial interactions. Carbohydrate binding modules (CBM) are often associated with microbial surface-tethered cellulosomal or freely secreted cellulase enzymes to increase substrate accessibility. However, it is not well known how CBM recognize, bind, and dissociate from polysaccharide surfaces to facilitate efficient cellulolytic activity due to the lack of mechanistic understanding of CBM-substrate interactions. Our work outlines a general approach to methodically study the unbinding behavior of CBMs from model polysaccharide surfaces using single-molecule force spectroscopy. Here, we apply acoustic force spectroscopy (AFS) to probe a Clostridium thermocellum cellulosomal scaffoldin protein (CBM3a) and measure its dissociation from nanocellulose surfaces at physiologically relevant, low force loading rates. An automated microfluidic setup and methodology for uniform deposition of insoluble polysaccharides on the AFS chip surfaces is demonstrated. The rupture forces of wild-type CBM3a, and its Y67A mutant, unbinding from nanocellulose surface suggests distinct CBM binding conformations that can also explain the improved cellulolytic activity of cellulase tethered to CBM. Applying established dynamic force spectroscopy theory, the single-molecule unbinding rate at zero force is extrapolated and found to agree well with bulk equilibrium unbinding rates estimated independently using quartz crystal microbalance with dissipation monitoring. However, our results highlight the limitations of applying classical theory to explain the highly multivalent CBM-cellulose interactions seen at higher cellulose-CBM bond rupture forces (>15pN).

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Conjugation of DNA with protein using His-tag chemistry and its application to the aptamer-based detection system

Cited by 30 publications

References 20 publications

Genetically Encoded Multispectral Labeling of Proteins with Polyfluorophores on a DNA Backbone

Genetically Encoded Multispectral Labeling of Proteins with Polyfluorophores on a DNA Backbone

Hexahistidine-tag-specific optical probes for analyses of proteins and their interactions

Acoustic Force Spectroscopy Reveals Subtle Differences in Cellulose Unbinding Behavior of Carbohydrate-Binding Modules

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