Site-specific protein labelling and immobilization mediated by microbial transglutaminase

Abstract

Microbial transglutaminase (mTG) shows broad substrate specificity that is amenable to in vitro bio-conjugation applications. Herein, test proteins were genetically fused with peptide tags, followed by mTG-mediated propargylation of their reactive Gln residues. The propargylated proteins were subjected to copper-assisted azide-alkyne cycloaddition to demonstrate either fluorescent labelling or immobilization.

Peptide-tags for site-specific protein labelling in vitro and in vivo

“…46 Recent studies described the use of tissue TGase (TG2) or microbial TGase (mTGase). 47,48 The latter provides the advantage of calcium-independent labelling. Transfer of propargylamine with subsequent CuAAC modification was demonstrated for maltose binding protein using mTGase.…”

A Green BODIPY‐Based, Super‐Fluorogenic, Protein‐Specific Labelling Agent

“…14–30 kDa) of these tags poses a similar risk for steric perturbation of the POI. 3) Alternatively, a “substrate tag” that is recognised by an enzyme can be fused to a POI . A site‐specific enzymatic reaction can then be used to affix a fluorescent label to the POI.…”

Engineered, highly reactive substrates of microbial transglutaminase enable protein labeling within various secondary structure elements

“…In contrast, MTG's glutamine reactivity is restricted to protein‐ and peptide‐bound glutamine residues. Phage display screening of glutamine‐containing peptides has yielded several “glutamine tags” that were successfully applied to channel MTG's reactivity during protein labeling; we note that this example used a C ‐terminally expressed glutamine recognition tag rather than a reactive glutamine internal to the target protein. Nonetheless, those glutamine‐containing sequences are diverse in composition, revealing no clear pattern in the primary structure surrounding the reactive glutamine …”

“…[5][6][7][8] One of the foundations of this approach is to optimize the incorporation efficiency of the label onto a protein of interest. MTG has been applied for fluorescent labeling [9][10][11] yet the determinants for its selective reactivity remains elusive. The deconvolution of these details holds great potential for improving MTG's labeling capacity.…”