Recombinant Expression of Cyclotides Using Split Inteins

Area of Science:

• Biochemistry
• Molecular Biology
• Plant Science

Background:

• Cyclotides are plant-derived microproteins characterized by a cyclic, head-to-tail arrangement and a distinctive knot topology stabilized by three disulfide bonds.
• These microproteins exhibit pharmacologically relevant activities and possess cell membrane permeability, highlighting their therapeutic potential.
• The unique structural features of cyclotides make them suitable scaffolds for grafting peptide epitopes, enabling the development of novel peptide-based therapeutics.

Purpose of the Study:

• To describe a method for expressing native folded cyclotides.
• To utilize intein-mediated protein trans-splicing for cyclotide expression in live Escherichia coli cells.
• To establish a system for producing cyclotides for potential therapeutic applications.

Main Methods:

• Intein-mediated protein trans-splicing was employed for cyclotide expression.
• The expression system was implemented in live Escherichia coli cells.
• The focus was on achieving native folding of the cyclotide structure.

Main Results:

• Successful expression of a native folded cyclotide was achieved.
• The intein-mediated protein trans-splicing method facilitated the production of correctly folded cyclotides.
• The study demonstrated the feasibility of using E. coli as a host for cyclotide expression.

Conclusions:

• Intein-mediated protein trans-splicing is an effective method for expressing native folded cyclotides in E. coli.
• This approach provides a valuable platform for the production of cyclotides for therapeutic development.
• The ability to express functional cyclotides opens avenues for molecular grafting and the design of novel peptide therapeutics.