A rapid, site-selective and efficient route to the dual modification of DARPins

Moody, Paul; Chudasama, Vijay; Nathani, Ramiz I.; Maruani, Antoine; Martin, Stephen M.; Smith, Mark E.; Caddick, Stephen

doi:10.1039/c4cc00053f

Cited by 17 publications

(19 citation statements)

References 33 publications

(37 reference statements)

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“…Two ubiquitin moieties were conjugated through haloalkyl substitution, through which a Michael addition acceptor (in this case an α,β‐unsaturated moiety) was attached for probing purposes to the active‐site cysteine residue in deubiquitinases . DARPins (designed ankyrin repeat proteins) can be dually modified at carefully positioned cysteine residues, as shown by Moody et al., who functionalised these binding proteins by sequential substitution and maleimide coupling …”

Section: Modification By Substitutionmentioning

confidence: 99%

Chemical Protein Modification through Cysteine

2016

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The modification of proteins with non-protein entities is important for a wealth of applications, and methods for chemically modifying proteins attract considerable attention. Generally, modification is desired at a single site to maintain homogeneity and to minimise loss of function. Though protein modification can be achieved by targeting some natural amino acid side chains, this often leads to ill-defined and randomly modified proteins. Amongst the natural amino acids, cysteine combines advantageous properties contributing to its suitability for site-selective modification, including a unique nucleophilicity, and a low natural abundance--both allowing chemo- and regioselectivity. Native cysteine residues can be targeted, or Cys can be introduced at a desired site in a protein by means of reliable genetic engineering techniques. This review on chemical protein modification through cysteine should appeal to those interested in modifying proteins for a range of applications.

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Section: Modification By Substitutionmentioning

confidence: 99%

Chemical Protein Modification through Cysteine

2016

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“…Given the lack of cysteines in their structure, it is possible to introduce a unique cysteine that is surface accessible for conjugation of a variety of molecules, such as drugs, proteins, imaging reagents, and nanomaterials, to the DARPin. The introduction of a unique cysteine into the DARPin structure also allows control of the orientation of conjugates to better diminish potential steric hindrance (148, 149). …”

Section: Antibody Mimeticsmentioning

confidence: 99%

Beyond Antibodies as Binding Partners: The Role of Antibody Mimetics in Bioanalysis

Yang

Dikici

et al. 2017

Annual Rev. Anal. Chem.

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The emergence of novel binding proteins or antibody mimetics capable of binding to ligand analytes in a manner analogous to that of the antigen–antibody interaction has spurred increased interest in the biotechnology and bioanalytical communities. The goal is to produce antibody mimetics designed to outperform antibodies with regard to binding affinities, cellular and tumor penetration, large-scale production, and temperature and pH stability. The generation of antibody mimetics with tailored characteristics involves the identification of a naturally occurring protein scaffold as a template that binds to a desired ligand. This scaffold is then engineered to create a superior binder by first creating a library that is then subjected to a series of selection steps. Antibody mimetics have been successfully used in the development of binding assays for the detection of analytes in biological samples, as well as in separation methods, cancer therapy, targeted drug delivery, and in vivo imaging. This review describes recent advances in the field of antibody mimetics and their applications in bioanalytical chemistry, specifically in diagnostics and other analytical methods.

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“…The main approaches to dually modify peptides and proteins can be classified in two separate categories (Figure 1). 44 The first one is the modification of two amino acids on the proteins using various methods that include: exploiting the difference of accessibility or reactivity of amino acids, [45][46][47] functionalisation of the C-and N-terminus of proteins, 48 use of unnatural amino acids, [49][50][51] use of specific enzymes 50 or a combination of these strategies 52 . The second category involves conjugation of a multifunctional linker to the protein of interest.…”

Section: Dual Modification Of Peptides and Proteinsmentioning

confidence: 99%