Residue choice defines efficiency and influence of bioorthogonal protein modification via genetically encoded strain promoted Click chemistry

Reddington, Samuel C.; Tippmann, Eric Michael; Jones, D. Dafydd

doi:10.1039/c2cc31887c

Cited by 50 publications

(66 citation statements)

References 25 publications

(26 reference statements)

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“…It has been reported that higher solvent-accessibility of a NNAA generally ensures higher conjugation efficiency [69–71]. However, it should be noted that NNAA substitution for an original amino acid with a fairly different structure and charge may undermine the accessibility-efficiency correlation [72]. Therefore, AzF incorporation sites, W160 and W174, were chosen out of amino acids bearing an aromatic ring, i.e., Phe, Trp, and Tyr, as well as high solvent accessibility.…”

Section: Discussionmentioning

confidence: 99%

Site-specific albumination of a therapeutic protein with multi-subunit to prolong activity in vivo

Lim

Hahn

Kwon

2015

Journal of Controlled Release

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Albumin fusion/conjugation (albumination) has been an effective method to prolong in vivo half-life of therapeutic proteins. However, its broader application to proteins with complex folding pathway or multi-subunit is restricted by incorrect folding, poor expression, heterogeneity, and loss of native activity of the proteins linked to albumin. We hypothesized that the site-specific conjugation of albumin to a permissive site of a target protein will expand the utilities of albumin as a therapeutic activity extender to proteins with a complex structure. We show here the genetic incorporation of a non-natural amino acid (NNAA) followed by chemoselective albumin conjugation to prolong therapeutic activity in vivo. Urate oxidase (Uox), a therapeutic enzyme for treatment of hyperuricemia, is a homotetramer with multiple surface lysines, limiting conventional approaches for albumination. Incorporation of p-azido-l-phenylalanine into two predetermined positions of Uox allowed site-specific linkage of dibenzocyclooctyne-derivatized human serum albumin (HSA) through strain-promoted azide-alkyne cycloaddition (SPAAC). The bio-orthogonality of SPAAC resulted in the production of a chemically well-defined conjugate, Uox-HSA, with a retained enzymatic activity. Uox-HSA had a half-life of 8.8 h in mice, while wild-type Uox had a half-life of 1.3 h. The AUC increased 5.5-fold (1657 vs. 303 mU/mL × h). These results clearly demonstrated that site-specific albumination led to the prolonged enzymatic activity of Uox in vivo. Site-specific albumination enabled by NNAA incorporation and orthogonal chemistry demonstrates its promise for the development of long-acting protein therapeutics with high potency and safety.

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Section: Discussionmentioning

confidence: 99%

Site-specific albumination of a therapeutic protein with multi-subunit to prolong activity in vivo

Lim

Hahn

Kwon

2015

Journal of Controlled Release

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“…The most highly reactive site ( k 2 = 17.8 M −1 s −1 ) had the least surface exposure of all tested sites and was in a non-charged but polar local environment with an adjacent aromatic phenylalanine residue. [22] …”

Section: Discussionmentioning

confidence: 99%

Micelle‐Enhanced Bioorthogonal Labeling of Genetically Encoded Azido Groups on the Lipid‐Embedded Surface of a GPCR

2015

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Genetically-encoded p-azido-phenylalanine (azF) residues in G protein-coupled receptors (GPCRs) can be targeted with dibenzocyclooctyne (DIBO) modified fluorescent probes using strain-promoted [3+2] azide–alkyne cycloaddition (SpAAC). Here we show that azF residues situated on the transmembrane surface of detergent-solubilized receptors exhibit up to 1000-fold rate enhancement compared with azF residues on water-exposed surfaces. We show that the amphipathic moment of the labeling reagent, consisting of hydrophobic DIBO coupled to hydrophilic Alexa dye, results in strong partitioning of the DIBO group into the hydrocarbon core of the detergent micelle and consequently high local reactant concentrations. The observed rate constant for the micelle-enhanced SpAAC is comparable with the fastest bioorthogonal labeling reactions known. Targeting hydrophobic regions of membrane proteins using the micelle-enhanced SpAAC reaction should expand the utility of bioorthogonal labeling strategies.

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“…[39] Interestingly, DIBO appeared to have drastically reduced background labeling as compared with BCN and DIBAC. [39] Previous applications of SpAAC for site-specific protein labeling primarily involved highly expressing, soluble proteins such as GFP expressed in E. coli and purified by Ni-NTA affinity chromatography followed by one of two alternative schemes: i) incorporation of azF into the target protein and subsequent labeling with DBCO (a cyclooctyne closely related to DIBAC), [40] or ii) incorporation of BCN derivative of pyrrolysine followed by labeling with an azido-containing label. [41] …”

Section: Discussionmentioning

confidence: 99%

“…[16a, 31b] A similar effect has been observed for the modification of GFP using a similar cyclooctyne reagent. [40] A straightforward explanation would be that the lipophilic nature of DIBO, [31b] which enables the labeling reagent to partition into the dodecyl maltoside micelles, results in an increased local concentration at sites located in the hydrophobic region of rhodopsin.…”

Section: Discussionmentioning

confidence: 99%

Bioorthogonal Fluorescent Labeling of Functional G‐Protein‐Coupled Receptors

et al. 2014

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Novel methods are required for site-specific, quantitative fluorescence labeling of G protein-coupled receptors (GPCRs) and other difficult-to-express membrane proteins. Ideally, fluorescent probes should perturb native structure and function as little as possible. We evaluated bioorthogonal reactions to label genetically encoded p-acetyl-l-phenylalanine (AcF) or p-azido-l-phenylalanine (azF) residues in receptors heterologously expressed in mammalian cells. We found that keto-selective reagents were not truly bioorthogonal, possibly due to posttranslational protein oxidation reactions. In contrast, the strain-promoted [3+2] azide–alkyne cycloaddition (SpAAC) with dibenzocyclooctyne (DIBO) reagents yielded stoichiometric conjugates with azF-rhodopsin while undergoing negligible background reactions. As one useful application, we used Alexa488-rhodopsin to measure the kinetics of ligand uptake and release in membrane-mimetic bicelles using a novel fluorescence-quenching assay.

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Residue choice defines efficiency and influence of bioorthogonal protein modification via genetically encoded strain promoted Click chemistry

Cited by 50 publications

References 25 publications

Site-specific albumination of a therapeutic protein with multi-subunit to prolong activity in vivo

Site-specific albumination of a therapeutic protein with multi-subunit to prolong activity in vivo

Micelle‐Enhanced Bioorthogonal Labeling of Genetically Encoded Azido Groups on the Lipid‐Embedded Surface of a GPCR

Bioorthogonal Fluorescent Labeling of Functional G‐Protein‐Coupled Receptors

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