An expanded library of orthogonal split inteins enables modular multi-peptide assemblies.

Area of Science:

• Biochemistry
• Synthetic Biology
• Protein Engineering

Background:

• Inteins are protein segments that catalyze protein splicing, a process joining flanking sequences without being present in the final product.
• This self-catalytic activity allows for scarless peptide ligation, a valuable tool in biotechnology.
• Developing a diverse and orthogonal set of inteins is crucial for advanced applications.

Purpose of the Study:

• To characterize the splicing activity of 34 inteins using a high-throughput platform.
• To establish a library of mutually orthogonal split inteins for versatile applications.
• To demonstrate the utility of these inteins in synthetic biology and protein engineering.

Main Methods:

• A rapid split fluorescent reporter platform was employed to assess the splicing activity of 34 inteins.
• Split inteins were engineered for mutual orthogonality, enabling their independent function.
• The functionality of orthogonal split inteins was tested in implementing logic circuits and assembling large proteins.

Main Results:

• A library of 15 mutually orthogonal split inteins was successfully established.
• Ten of these inteins demonstrated simultaneous utility in in vitro applications.
• Orthogonal split inteins were shown to facilitate complex logic circuits in vivo and seamless assembly of large proteins in vitro.

Conclusions:

• The expanded library of orthogonal split inteins offers significant versatility for synthetic biology and protein engineering.
• These inteins provide a powerful tool for scarless protein ligation and the construction of complex biomolecular systems.
• The developed platform and intein library pave the way for novel biotechnological advancements.