Modular construction of multi-subunit protein complexes using engineered tags and microbial transglutaminase

Area of Science:

• Synthetic Biology
• Biochemistry
• Materials Science

Background:

• Hierarchical assembly of protein complexes is crucial for applications in biosensing, biomedicine, and bioreactors.
• Current assembly methods often lack simplicity, scalability, and versatility, leading to component loss.
• A
• plug and play
• methodology is needed for efficient protein complex design and optimization.

Purpose of the Study:

• To develop a modular and scalable protein assembly method using a generic linking apparatus.
• To enable the construction of multifunctional protein complexes for applications like metabolon construction and molecular communication devices.
• To demonstrate the versatility of the proposed method in altering protein stoichiometries and positions.

Main Methods:

• Incorporation of crosslinking-compliant amino acid tags (lysine or glutamine) at protein termini.
• Covalent crosslinking of tagged proteins using microbial transglutaminase (mTG).
• Demonstration of modularity by altering enzyme stoichiometries and positions, and construction of multifunctional complexes.

Main Results:

• Successful construction of a two-subunit quorum sensing (QS) biosynthetic metabolon on solid supports.
• Increased reaction rates by altering stoichiometries of limiting constituents.
• Development of a molecular communication device (antibody binding Protein G-QS complex) for targeted bacterial cell interaction and tailored QS responses.

Conclusions:

• The solid-phase mTG-mediated protein linkage approach offers a general and versatile method for constructing protein complexes.
• This methodology facilitates a
• plug and play
• assembly of multifunctional protein complexes for diverse biosensing and synthetic biology applications.
• The approach is suitable for various environments, including microreactors and lab-on-a-chip systems.