Labeling proteins on live mammalian cells using click chemistry

Abstract

We describe a protocol for the rapid labeling of cell-surface proteins in living mammalian cells using click chemistry. The labeling method is based on strain-promoted alkyne-azide cycloaddition (SPAAC) and strain-promoted inverse-electron-demand Diels-Alder cycloaddition (SPIEDAC) reactions, in which noncanonical amino acids (ncAAs) bearing ring-strained alkynes or alkenes react, respectively, with dyes containing azide or tetrazine groups. To introduce ncAAs site specifically into a protein of interest (POI), we use genetic code expansion technology. The protocol can be described as comprising two steps. In the first step, an Amber stop codon is introduced--by site-directed mutagenesis--at the desired site on the gene encoding the POI. This plasmid is then transfected into mammalian cells, along with another plasmid that encodes an aminoacyl-tRNA synthetase/tRNA (RS/tRNA) pair that is orthogonal to the host's translational machinery. In the presence of the ncAA, the orthogonal RS/tRNA pair specifically suppresses the Amber codon by incorporating the ncAA into the polypeptide chain of the POI. In the second step, the expressed POI is labeled with a suitably reactive dye derivative that is directly supplied to the growth medium. We provide a detailed protocol for using commercially available ncAAs and dyes for labeling the insulin receptor, and we discuss the optimal surface-labeling conditions and the limitations of labeling living mammalian cells. The protocol involves an initial cloning step that can take 4-7 d, followed by the described transfections and labeling reaction steps, which can take 3-4 d.

Molecular basis for functional switching of GFP by two disparate non-native post-translational modifications of a phenyl azide reaction handle

“…1,47,48 However, little is known about the impact on protein structure and how any induced structural changes can be used to influence function, in a manner akin to natural PTM events. SPAAC is generally used to attach labels to proteins with no anticipated changes in structure and function but recent studies have indicated it can be used to control enzyme activity, presumably through active site blocking.…”

Site-Specific Bioorthogonal Labeling for Fluorescence Imaging of Intracellular Proteins in Living Cells

“…70,90 Nevertheless, our labeling and imaging results as well as studies from others 71 suggest that the ligation product is still relatively stable and dominant, perhaps due to conjugation of the bulky fluorophores. Interestingly, due to the higher reactivity of exo-BCNK with tetrazines than 2’-aTCOK, 48,55 the relative fluorescence labeling efficiency of exo-BCNK after normalization with protein expression level is generally higher than 2’-aTCOK. Therefore, when low protein expression level is desired, exo-BCNK may be used, although the overall fluorescence signal might be dimmer under fluorescence microscopy (Figure S24 and S25).…”

“…6,48 For instance, the UAA should be stable for prolonged incubation time in cell culture medium under physiological temperature, and should be highly efficiently incorporated into proteins of interest in mammalian cells. In addition, the organic fluorophores should be carefully selected, as their cellular properties, such as membrane permeability, intracellular distribution, and retention, can affect protein labeling efficiency and specificity.…”

The use of unnatural amino acids to study and engineer protein function

“…strained alkenes or alkynes) and fluorophores with corresponding reactivity provides a powerful alternative to conventional fluorophore tagging methods [70][71][72]. However, the approach is technically demanding because it requires sophisticated protocols for the removal of unreacted dye [73]. Ideally the fluorescence of the dye is activated by the coupling reaction; as it is the case for some azido-coumarins and tetrazine-fluorophores [70][71][72].…”