Enhancing substrate specificity of microbial transglutaminase for precise nanobody labeling

Area of Science:

• Biochemistry
• Molecular Biology
• Protein Engineering

Background:

• Streptomyces mobaraenesis transglutaminase (smTG) is utilized for protein labeling.
• Existing smTG methods can lead to non-specific protein cross-linking.
• Precise site-specific labeling is crucial for applications like antibody-drug conjugates.

Purpose of the Study:

• To engineer smTG for precise site-specific labeling of proteins.
• To develop a method for creating nanobody-fluorophore conjugates (NFCs).
• To enhance smTG activity towards a specific peptide substrate for targeted conjugation.

Main Methods:

• Utilized molecular docking and virtual mutagenesis to redesign smTG substrate specificity.
• Introduced single point mutations (G250H, Y278E) into a thermostable smTG variant (TGm2).
• Assessed enzyme activity against the GGGGQR peptide and standard substrates.
• Demonstrated site-specific labeling of nanobodies (1C12, 7D12) with a fluorophore using engineered smTG.

Main Results:

• Mutations G250H and Y278E significantly enhanced smTG activity against the GGGGQR peptide.
• The Y278E mutation dramatically shifted substrate preference, increasing GGGGQR activity ratio from 0.05 to 0.93.
• Engineered smTG variant TGm2-Y278E achieved successful site-specific labeling of nanobodies fused with GGGGQR.
• Non-specific labeling was observed with other smTG variants, highlighting the specificity of TGm2-Y278E.

Conclusions:

• Engineering smTG via targeted mutations can achieve precise site-specific protein labeling.
• Modified smTG variants are effective tools for the biosynthesis of nanobody-fluorophore conjugates.
• This approach holds promise for the development of antibody-drug conjugates and other bioconjugates.