Choosing an effective protein bioconjugation strategy

Abstract

The collection of chemical techniques that can be used to attach synthetic groups to proteins has expanded substantially in recent years. Each of these approaches allows new protein targets to be addressed, leading to advances in biological understanding, new protein-drug conjugates, targeted medical imaging agents and hybrid materials with complex functions. The protein modification reactions in current use vary widely in their inherent site selectivity, overall yields and functional group compatibility. Some are more amenable to large-scale bioconjugate production, and a number of techniques can be used to label a single protein in a complex biological mixture. This review examines the way in which experimental circumstances influence one's selection of an appropriate protein modification strategy. It also provides a simple decision tree that can narrow down the possibilities in many instances. The review concludes with example studies that examine how this decision process has been applied in different contexts.

Bringing single-molecule spectroscopy to macromolecular protein complexes

“…It was anticipated to combine this approach with single molecule techniques and observe the assembly and function of macromolecular complexes in real time. However, technical hurdles related to the specificity and efficiency of labeling molecules of interest within crude cell extracts had been reported [22][23][24]. In order to overcome this limitation, several groups recently developed original approaches.…”

“…A variety of chemical strategies are used for protein labeling [22][23][24]. These approaches can be reliably practiced with purified proteins, but many of these schemes suffer from an undesirably low quality of conjugation when they are performed intracellularly or within crude cell extracts.…”

“…These techniques involve the recognition of specific amino-acid motifs of cellular proteins that are then modified with remarkably high specificity. Detailed descriptions of the protein-directed and protein-mediated labeling strategies can be found in other reviews [22][23][24].…”

“…Due to this limitation, the use of cell extracts represents a promising alternative that has been successfully adopted to perform in vitro splicing of labeled pre-mRNA at the singlemolecule level [26,27]. Although it was anticipated that the kinetics of the assembly and action of the spliceosome would be discovered soon after the development and use of single- A variety of chemical strategies are used for protein labeling [22][23][24]. These approaches can be reliably practiced with purified proteins, but many of these schemes suffer from an undesirably low quality of conjugation when they are performed intracellularly or within crude cell extracts.…”

Transition Metal-Promoted Arylation: An Emerging Strategy for Protein Bioconjugation

“…[3] Although nature is readily able to modify complex peptides and proteins using highly specific enzyme-catalyzed transformations, termed post-translational modifications (PTMs), [4] duplicating the mild reaction conditions (physiological pH, aqueous media, and ambient temperature) and the exquisite chemo-and regioselectivity of enzymatic transformations in the laboratory is an on-going challenge. [5] The majority of classical methods for bioconjugation rely on the modification of select proteinogenic amino acid residues, generally exploiting the native nucleophilicity of lysine (Lys), tyrosine (Tyr), and cysteine (Cys) side chain functionalities. [1b,6] Unnatural amino acids, incorporated using chemical synthesis or biological expression, have enabled the introduction of strategic non-native functional handles (e.g.…”