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Tagging and Fusion Proteins

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Site-specific protein propargylation using tissue transglutaminase.

Claudio Gnaccarini1, Wajih Ben-Tahar, Amina Mulani

  • 1Département de Chimie, Université de Montreal, Succursale centre-ville, C.P. 6128, Montréal, Québec H3C 3J7, Canada.

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Researchers developed a new assay to detect transglutaminase 2 (TG2) activity, identifying propargyl amine as a novel substrate. This enables site-specific protein modification using TG2 and bioorthogonal chemistry.

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Area of Science:

  • Biochemistry
  • Enzymology
  • Chemical Biology

Background:

  • Transglutaminases (TGases) are enzymes catalyzing transamidation reactions involving glutamine residues.
  • Tissue transglutaminase 2 (TG2) plays roles in various cellular processes, including cross-linking and signaling.
  • Developing specific assays for TG2 activity is crucial for understanding its function and for biotechnological applications.

Purpose of the Study:

  • To develop a novel FRET-based assay for directly detecting TG2-mediated ligation reactions.
  • To screen for novel amine substrates of TG2.
  • To demonstrate the utility of TG2 in site-specific protein modification using bioorthogonal chemistry.

Main Methods:

  • Development of a Förster Resonance Energy Transfer (FRET)-based activity assay for TG2.
  • Microtiter plate-based screening of 18 potential amine substrates.
  • Kinetic characterization (K(M) determination) of identified TG2 substrates.
  • Proof-of-principle protein labeling using TG2 and subsequent bioorthogonal functionalization.

Main Results:

  • The novel FRET assay successfully detected TG2-mediated ligation.
  • Propargyl amine was identified as an excellent substrate for TG2.
  • Kinetic parameters for propargyl amine (K(M) = 44 ± 4 μM) and 2-azidoethyl amine (K(M) = 0.99 ± 0.06 mM) were determined.
  • Site-specific labeling of casein was achieved using TG2 and propargyl amine, followed by fluorescent tagging via click chemistry.

Conclusions:

  • A novel FRET assay provides a direct method for measuring TG2 activity.
  • Propargyl amine is a highly effective substrate for TG2, enabling new chemical modifications.
  • TG2, in conjunction with propargyl amine and bioorthogonal chemistry, offers a powerful tool for site-specific protein functionalization.