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

Abstract

Microbial transglutaminase (MTG) is a practical tool to enzymatically form isopeptide bonds between peptide or protein substrates. This natural approach to crosslinking the side-chains of reactive glutamine and lysine residues is solidly rooted in food and textile processing. More recently, MTG's tolerance for various primary amines in lieu of lysine have revealed its potential for site-specific protein labeling with aminated compounds, including fluorophores. Importantly, MTG can label glutamines at accessible positions in the body of a target protein, setting it apart from most labeling enzymes that react exclusively at protein termini. To expand its applicability as a labeling tool, we engineered the B1 domain of Protein G (GB1) to probe the selectivity and enhance the reactivity of MTG toward its glutamine substrate. We built a GB1 library where each variant contained a single glutamine at positions covering all secondary structure elements. The most reactive and selective variants displayed a >100-fold increase in incorporation of a recently developed aminated benzo[a]imidazo[2,1,5-cd]indolizine-type fluorophore, relative to native GB1. None of the variants were destabilized. Our results demonstrate that MTG can react readily with glutamines in α-helical, β-sheet, and unstructured loop elements and does not favor one type of secondary structure. Introducing point mutations within MTG's active site further increased reactivity toward the most reactive substrate variant, I6Q-GB1, enhancing MTG's capacity to fluorescently label an engineered, highly reactive glutamine substrate. This work demonstrates that MTG-reactive glutamines can be readily introduced into a protein domain for fluorescent labeling.

RNA‐dependent chaperone (chaperna) as an engineered pro‐region for the folding of recombinant microbial transglutaminase

“…Particularly, microbial TGase has been utilized in the processing of food products containing meat, fish, dairy, and wheat (Buchert et al, ; Motoki & Seguro, ). Moreover, TGase has been utilized in nonfood industries, such as protein engineering, textile and leather processing, pharmaceutical industries, and development of surgical materials (Fontana, Spolaore, Mero, & Veronese, ; Y. Liu et al, ; Rachel, Quaglia, Levesque, Charette, & Pelletier, ; Zhu & Tramper, ). Among the many microbial TGases that have been studied for their expression and activity in Escherichia coli , Streptomyces mobaraensis TGase (Sm TGase) is the first industrial enzyme (Kawai, Takehana, & Takagi, ; Pasternack et al, ; Yokoyama, Nakamura, Seguro, & Kubota, ).…”

Incorporation of TePhe into Expressed Proteins is Minimally Perturbing

“…Optimization of conditions for metabolic incorporation of TePhe into His 6 -GB1 in E. coli N-terminally his-tagged GB1 (His 6 -GB1) was expressed from a pET15b plasmid (generous gift of Prof. Joelle Pelletier) [28] in BL21 (DE3) E. coli. This construct contains a thrombin site following the his-tag which when cut leaves a Gly-Ser-His leader prior the start methionine of GB1 (see Supporting Information for full sequence).…”

Site-Specific Conjugation of Metal-Chelating Polymers to Anti-Frizzled-2 Antibodies via Microbial Transglutaminase

“…The enzyme catalyzes an acyl transfer reaction at certain glutamine side chains, where the acyl group can be transferred to various primary amines for conjugation or to water in a competing hydrolysis reaction. The exact sequence and structural requirements for the glutamine substrate are still under investigation. − In terms of the amine substrate, it has been reported that the enzyme poorly tolerates negatively charged substrates including negatively charged polymers, where the enzyme failed to conjugate an amine-terminated oligonucleotide to a classic acyl donor model protein N , N -dimethyl casein. However, the enzyme has been used for modifying a variety of proteins including antibodies and for polymers such as PEG .…”