Biosynthesis of the Cyclotide Kalata B1 using a Protein Splicing Unit
Cyclotides are a newly emerging family of large backbone cyclic polypeptides ({approx}30 residues long) characterized by a disulfide-stabilized core (3 disulfide bonds) with an unusual knotted structure. In contrast to other cyclic polypeptides, cyclotides have a well-defined three-dimensional structure. Therefore, despite their small size, they can be considered miniproteins. The unique cyclic-backbone topology and knotted arrangement of 3 disulfide bonds endow cyclotides with exceptional stability and resistance to chemical, enzymatic and thermal degradation. Furthermore, their well-defined structures have been associated with a range of biological functions. Together, these characteristics suggest that cyclotides are ideal molecular scaffolds for the development of stable peptide drugs. Despite the fact that the chemical synthesis of circular peptides has been well explored and a number different approaches involving solid-phase or liquid-phase exist, recent developments in the fields of molecular biology and protein engineering have now made possible the biosynthesis of cyclic peptides. This progress has been made mainly in two areas, non-ribosomal peptide synthesis and Expressed Protein Ligation (EPL)/protein trans-splicing. Access to biosynthetic cyclotides using recombinant DNA expression techniques offers the exciting possibility of producing large combinatorial libraries of highly stable miniproteins. This would allow the generation of cell-based combinatorial libraries that could be screened either in vitro or in vivo for their ability to regulate cellular processes. In the present work, we describe the biosynthesis of the cyclotide Kalata B1 (KB1) in E. coli using an engineered intein. Our approach (Figure 1) is based on an intramolecular version of Native Chemical Ligation (NCL). NCL involves the chemoselective reaction between a N-terminal Cys residue of one peptide and an {alpha}-thioester group of a second peptide. Importantly, incorporation of these two groups into the same synthetic polypeptide leads to efficient circularization.
- Research Organization:
- Lawrence Livermore National Lab. (LLNL), Livermore, CA (United States)
- Sponsoring Organization:
- USDOE
- DOE Contract Number:
- W-7405-ENG-48
- OSTI ID:
- 883753
- Report Number(s):
- UCRL-JRNL-214692; TRN: US200615%%214
- Journal Information:
- Angewandte Chemie International Edition, Vol. 45, Issue 6
- Country of Publication:
- United States
- Language:
- English
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