Synthesis and Application of a N-1′ Fluorescent Biotinyl Derivative Inducing the Specific Carboxy-Terminal Dual Labeling of a Novel RhoB-Selective scFv

Chaisemartin, L. De; Chinestra, Patrick; Favre, Gilles; Blonski, Casimir; Faye, Jean-Charles

doi:10.1021/bc800272r

Cited by 8 publications

(7 citation statements)

References 43 publications

(67 reference statements)

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“…Historically, the labeling of scFv was performed using fusion tags, providing the sites for the chemical reaction [ 40 , 41 ], because it was not certain whether the cysteine residue-dependent site-specific conjugation chemistry developed for the attachment of the full IgG molecule to scFv is applicable to scFv. To achieve this, the residues replaceable with cysteine without hindering the binding activity or solubility of scFv should be screened out and the binding activity of mutant scFv should not be affected by the conjugation process.…”

Section: Discussionmentioning

confidence: 99%

Chicken scFvs with an Artificial Cysteine for Site-Directed Conjugation

et al. 2016

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For the site-directed conjugation of chemicals and radioisotopes to the chicken-derived single-chain variable fragment (scFv), we investigated amino acid residues replaceable with cysteine. By replacing each amino acid of the 157 chicken variable region framework residues (FR, 82 residues on VH and 75 on VL) with cysteine, 157 artificial cysteine mutants were generated and characterized. At least 27 residues on VL and 37 on VH could be replaced with cysteine while retaining the binding activity of the original scFv. We prepared three VL (L5, L6 and L7) and two VH (H13 and H16) mutants as scFv-Ckappa fusion proteins and showed that PEG-conjugation to the sulfhydryl group of the artificial cysteine was achievable in all five mutants. Because the charge around the cysteine residue affects the in vivo stability of thiol-maleimide conjugation, we prepared 16 charge-variant artificial cysteine mutants by replacing the flanking residues of H13 with charged amino acids and determined that the binding activity was not affected in any of the mutants except one. We prepared four charge-variant H13 artificial cysteine mutants (RCK, DCE, ECD and ECE) as scFv-Ckappa fusion proteins and confirmed that the reactivity of the sulfhydryl group on cysteine is active and their binding activity is retained after the conjugation process.

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Section: Discussionmentioning

confidence: 99%

Chicken scFvs with an Artificial Cysteine for Site-Directed Conjugation

et al. 2016

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“…In addition, the general approaches employing EPL chemistries described here could also be used for covalent introduction of numerous other useful chemical functionalities. 20,46 …”

Section: Discussionmentioning

confidence: 99%

Facile Chemical Functionalization of Proteins through Intein-Linked Yeast Display

et al. 2013

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Intein-mediated expressed protein ligation (EPL) permits the site-specific chemical customization of proteins. While traditional techniques have used purified, soluble proteins, we have extended these methods to release and modify intein fusion proteins expressed on the yeast surface, thereby eliminating the need for soluble protein expression and purification. To this end, we sought to simultaneously release yeast surface-displayed proteins and selectively conjugate with chemical functionalities compatible with EPL and click chemistry. Single-chain antibodies (scFv) and green fluorescent protein (GFP) were displayed on the yeast surface as fusions to the N-terminus of the Mxe GyrA intein. ScFv and GFP were released from the yeast surface with either a sulfur nucleophile (MESNA) or a nitrogen nucleophile (hydrazine) linked to an azido group. The hydrazine azide permitted the simultaneous release and azido functionalization of displayed proteins, but nonspecific reactions with other yeast proteins were detected, and cleavage efficiency was limited. In contrast, MESNA released significantly more protein from the yeast surface while also generating a unique thioester at the carboxy-terminus of the released protein. These protein thioesters were subsequently reacted with a cysteine alkyne in an EPL reaction and then employed in an azide–alkyne cycloaddition to immobilize the scFv and GFP on an azide-decorated surface with >90% site-specificity. Importantly, the immobilized proteins retained their activity. Since yeast surface display is also a protein engineering platform, these approaches provide a particularly powerful tool for the rapid assessment of engineered proteins.

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“…7 for a detailed schematic). Following a very similar approach, an anti-RhoB scFv was engineered with the GyrA intein at its C-terminus and subsequently modified with a cysteine-biotin-fluorescein construct [36]. While these strategies are elegant, their inherent complexity may ultimately limit their wide-scale use.…”

Section: Peptide Tagsmentioning

confidence: 99%

Site-Specifically Labeled Immunoconjugates for Molecular Imaging—Part 2: Peptide Tags and Unnatural Amino Acids

et al. 2016

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Molecular imaging using radioisotope- or fluorophore-labeled antibodies is increasingly becoming a critical component of modern precision medicine. Yet despite this promise, the vast majority of these immunoconjugates are synthesized via the random coupling of amine-reactive bifunctional probes to lysines within the antibody, a process that can result in heterogeneous and poorly defined constructs with suboptimal pharmacological properties. In an effort to circumvent these issues, the last 5 years have played witness to a great deal of research focused on the creation of effective strategies for the site-specific attachment of payloads to antibodies. These chemoselective modification methods yield immunoconjugates that are more homogenous and better defined than constructs created using traditional synthetic approaches. Moreover, site-specifically labeled immunoconjugates have also been shown to exhibit superior in vivo behavior compared to their randomly modified cousins. The over-arching goal of this two-part review is to provide a broad yet detailed account of the various site-specific bioconjugation approaches that have been used to create immunoconjugates for positron emission tomography (PET), single photon emission computed tomography (SPECT), and fluorescence imaging. In Part 1, we covered site-specific bioconjugation techniques based on the modification of cysteine residues and the chemoenzymatic manipulation of glycans. In Part 2, we will detail two families of bioconjugation approaches that leverage biochemical tools to achieve site-specificity. First, we will discuss modification methods that employ peptide tags either as sites for enzyme-catalyzed ligations or as radiometal coordination architectures. And second, we will examine bioconjugation strategies predicated on the incorporation of unnatural or non-canonical amino acids into antibodies via genetic engineering. Finally, we will compare the advantages and disadvantages of the modification strategies covered in both parts of the review and offer a brief discussion of the overall direction of the field.

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Synthesis and Application of a N-1′ Fluorescent Biotinyl Derivative Inducing the Specific Carboxy-Terminal Dual Labeling of a Novel RhoB-Selective scFv

Cited by 8 publications

References 43 publications

Chicken scFvs with an Artificial Cysteine for Site-Directed Conjugation

Chicken scFvs with an Artificial Cysteine for Site-Directed Conjugation

Facile Chemical Functionalization of Proteins through Intein-Linked Yeast Display

Site-Specifically Labeled Immunoconjugates for Molecular Imaging—Part 2: Peptide Tags and Unnatural Amino Acids

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