Site-specific, covalent immobilization of BirA by microbial transglutaminase: A reusable biocatalyst for in vitro biotinylation

dCas9-targeted locus-specific protein isolation method identifies histone gene regulators

“…Although our analysis of the D. melanogaster HisC takes advantage of the HisC's intrinsic repetitiveness to increase the effective target concentration, single-locus chromatin purification and identification of regulatory proteins have recently been reported (19,22). We believe that, in the future, improvements of CLASP by coupling with in vitro biotinylation of an attached tag by recombinant BirA could provide a powerful approach to probe multiple single-copy genomic targets to identify interesting regulatory proteins (55). .…”

Tag-mediated single-step purification and immobilization of recombinant proteins toward protein-engineered advanced materials

“…Microbial transglutaminases (MTGs), which catalyze the formation of a covalent bond between a free amine group and the side chain of a glutamine residue on one protein or peptide, have also been used to mediate the oriented and covalent immobilization of Q6-tagged enzymes onto solid substrates/supports ( Table 2 ) [93] . New highly reactive Q-tags for the Bacillus subtilis MTG ( Table 1 ) [94] and new MTGs with high substrate specificity [95] have been recently reported, providing new tools for MTG-mediated covalent immobilization of recombinant proteins, biomaterial formation and site-specific protein modification.…”

“…The functionalization of the surface of iron oxide MNPs with immobilized nickel (II) ions (Ni 2+ ) – an IMAC adaptation – is commonly used to achieve rapid and efficient purification (in terms of binding rate) of His-tagged proteins and also to immobilize a variety of biomolecules in a fast and cost-effective way ( Table 2 ) [31] , [42] , [119] . Similarly, silica-coated MNPs functionalized with nickel (II) ions [120] or modified with functional groups such as amine [93] or even with thiol [121] groups, are also commonly used for enzyme immobilization ( Table 2 ). Additionally, silica and graphene-based nanoparticles without a magnetic core and functionalized with nickel (II) ions [40] or free amine group [91] were also successfully employed to enhance immunoaffinity-based detection and purification, and to immobilize proteins without activity loss, thus demonstrating the wide applicability range of nanoparticles [24] , [122] .…”

Site-Selective Protein Modification: From Functionalized Proteins to Functional Biomaterials

“…In one example, biotin ligase with an N-terminal Q-tag was immobilized onto magnetic microspheres and was shown to retain >95% activity. 118 Newly discovered microbial transglutaminases, such as that from Kutzneria albida, show high specificity for their substrates (YRYRQ and RYESK) and have been used for the site-specific labeling of antibodies with biotin. 119 Hydrogel biomaterials have also been formed through transglutaminase-mediated crosslinking.…”