Phage-Selected Clickable Gln-Donor Peptide for Lys-Selective Fab Labeling Using Engineered Microbial Transglutaminase

Area of Science:

• Bioconjugation Chemistry
• Protein Engineering
• Enzymatic Modification

Background:

• Microbial transglutaminase (MTG) shows promise for antibody modification, particularly for antibody-drug conjugates (ADCs).
• Selective modification of lysine residues on native antibodies using MTG is difficult due to low intrinsic reactivity.
• Enzyme-antibody proximity and substrate screening are key to overcoming these challenges.

Purpose of the Study:

• To develop a method for site-selective modification of lysine residues on antibody surfaces using MTG.
• To identify and design highly reactive Gln-donor peptide substrates for MTG-mediated labeling.
• To create fluorescent Fab conjugates through efficient site-selective modification and click chemistry.

Main Methods:

• Screening a phage-displayed peptide library to identify reactive Gln-donor substrates.
• Designing an azide-functionalized Gln-donor peptide for click chemistry.
• Utilizing an engineered MTG-protein G fusion (EzMTG-pG) for Lys65-selective modification of Fab fragments.

Main Results:

• Identified reactive Gln-donor peptide substrates from a random peptide library.
• Successfully designed an azide-functionalized peptide substrate for click chemistry.
• Achieved efficient Lys65-selective modification of Fab fragments using EzMTG-pG and subsequent click chemistry to create fluorescent conjugates.

Conclusions:

• Developed practical guidelines for designing substrates for MTG-mediated site-selective protein modification.
• Demonstrated a novel strategy for efficient and selective antibody labeling.
• Opened new avenues for the development of antibody-drug conjugates and other bioconjugates.