Dodecin as carrier protein for immunizations and bioengineering applications

Area of Science:

• Bioengineering
• Protein Engineering
• Immunology

Background:

• Scaffold proteins are crucial in bioengineering for molecular recruitment and pathway enhancement.
• They can convert non-immunogenic molecules into immunogens when acting as carriers.
• Developing novel, stable scaffold proteins is essential for advancing these applications.

Purpose of the Study:

• To introduce and characterize the dodecin from Mycobacterium tuberculosis (mtDod) as a new scaffold protein.
• To demonstrate mtDod's utility for cargo attachment and its potential in antibody production.
• To evaluate mtDod's recombinant production and its effectiveness as a recruitment hub.

Main Methods:

• Recombinant production of mtDod in Escherichia coli.
• Engineering mtDod with domains for direct, non-covalent, and covalent cargo loading.
• Construction and purification of large mtDod-protein fusions (e.g., with superfolder green fluorescent protein).
• Utilizing mtDod with SYNZIP and SpyCatcher domains for post-translational cargo recruitment.
• Generating antibodies against target proteins using mtDod-peptide fusions.

Main Results:

• MtDod was successfully produced recombinantly in high yield and quality.
• Engineered mtDod variants facilitated various cargo loading strategies.
• Large protein fusions (up to 437 kDa) were purified, confirming mtDod's role as a recruitment hub.
• The mtDod/SpyCatcher system proved effective for post-translational cargo recruitment.
• Antibodies against human heat shock proteins and CHIP were successfully produced using mtDod-peptide fusions.

Conclusions:

• MtDod is a highly stable, spherical, and robust scaffold protein suitable for diverse bioengineering applications.
• Its engineered forms allow for efficient cargo attachment and recruitment, including large proteins.
• The mtDod system, particularly with SpyCatcher, offers a versatile platform for antibody generation and other biotechnological uses.