A Facile Method for Reversibly Linking a Recombinant Protein to DNA

Goodman, Rachel; Erben, Christoph; Malo, Jonathan; Ho, Wei M.; McKee, Mireya L.; Kapanidis, Achillefs N.; Turberfield, Andrew J.

doi:10.1002/cbic.200900165

Cited by 70 publications

(65 citation statements)

References 47 publications

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“…NTA 3 -tagged 48mer DNA was synthesized from triaminated DNA (IDT, Leuven, Belgium) according to the protocol of Goodman et al 39; the detailed procedure is described elsewhere24. Before each Nickel loading step, other divalent ions were removed by flowing a 5-mM EDTA solution in T-buffer over the sample for 10 min.…”

Section: Methodsmentioning

confidence: 99%

Protein analysis by time-resolved measurements with an electro-switchable DNA chip

et al. 2013

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Measurements in stationary or mobile phases are fundamental principles in protein analysis. Although the immobilization of molecules on solid supports allows for the parallel analysis of interactions, properties like size or shape are usually inferred from the molecular mobility under the influence of external forces. However, as these principles are mutually exclusive, a comprehensive characterization of proteins usually involves a multi-step workflow. Here we show how these measurement modalities can be reconciled by tethering proteins to a surface via dynamically actuated nanolevers. Short DNA strands, which are switched by alternating electric fields, are employed as capture probes to bind target proteins. By swaying the proteins over nanometre amplitudes and comparing their motional dynamics to a theoretical model, the protein diameter can be quantified with Angström accuracy. Alterations in the tertiary protein structure (folding) and conformational changes are readily detected, and even post-translational modifications are revealed by time-resolved molecular dynamics measurements.

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Section: Methodsmentioning

confidence: 99%

Protein analysis by time-resolved measurements with an electro-switchable DNA chip

et al. 2013

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“…9–13 Different strategies have been used to connect proteins to ODNs, using both noncovalent and covalent chemistries. Synthesizing discrete protein-ODN conjugates via noncovalent (e.g., biotin-streptavidin 5 and nickel-histidine 14 ) interactions is convenient, but the resulting conjugates can reversibly dissociate during purification and subsequent handling. Covalent protein-ODN conjugates have also been synthesized by connecting appropriately functionalized ODNs and proteins, using a wide variety of chemistries.…”

Section: Introductionmentioning

confidence: 99%

Covalent protein–oligonucleotide conjugates by copper-free click reaction

Khatwani

Kang

Mullen

et al. 2012

Bioorganic & Medicinal Chemistry

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Covalent protein-oligodeoxynucleotide (protein-ODN) conjugates are useful in a number of biological applications, but synthesizing discrete conjugates—where the connection between the two components is at a defined location in both the protein and the ODN—under mild conditions with significant yield can be a challenge. In this article, we demonstrate a strategy for synthesizing discrete protein-ODN conjugates using strain-promoted azide-alkyne [3+2] cycloaddition (SPAAC, a copper-free “click” reaction). Azide-functionalized proteins, prepared by enzymatic prenylation of C-terminal CVIA tags with synthetic azidoprenyl diphosphates, were “clicked” to ODNs that had been modified with a strained dibenzocyclooctyne (DIBO-ODN). The resulting protein-ODN conjugates were purified and characterized by size-exclusion chromatography and gel electrophoresis. We find that the yields and reaction times of the SPAAC bioconjugation reactions are comparable to those previously reported for copper-catalyzed azide-alkyne [3+2] cycloaddition (CuAAC) bioconjugation, but require no catalyst. The same SPAAC chemistry was used to immobilize azide-modified proteins onto surfaces, using surface-bound DIBO-ODN as a heterobifunctional linker. Cu-free click bioconjugation of proteins to ODNs is a simple and versatile alternative to Cu-catalyzed click methods.

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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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A Facile Method for Reversibly Linking a Recombinant Protein to DNA

Cited by 70 publications

References 47 publications

Protein analysis by time-resolved measurements with an electro-switchable DNA chip

Protein analysis by time-resolved measurements with an electro-switchable DNA chip

Covalent protein–oligonucleotide conjugates by copper-free click reaction

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

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