The KNOTTIN database

Chemical synthesis & recombinant protein expression

  • Since knottins are considered as interesting leads or scaffolds in drug design, it is necessary that these miniproteins can be obtained easily.
  • As they are very small proteins, many knottins were obtained by chemical synthesis.
  • Several linear and cyclic knottins were also obtained using recombinant technologies.
  • Main techniques used to prepare knottins (including cyclic knottins) are outlined below.

Knottins can be obtained either

Synthesis

Expression

Protease inhibitors

Cyclotides

Toxins

Others

Cyclotides

Synthesis of Bracelet cyclotide Cycloviolacin O2

The oxidative folding conditions used for kalata B1 appeared to produce only low yields of Bracelet cyclotides, i.e. cyclotides without the cis-Proline found in loop 'e' of Moebius cyclotides. A new improved strategy for the synthesis of Bracelet cycloviolacin O2 has been reported which uses Fmoc instead of BOC peptide chemistry and optimized conditions for the oxidative folding step (detergents, redox reagents, DMSO) [Leta Aboye et al., 2008].

15N labeled cyclotides

Complete labeling of cyclotides has been obtained by growing the cyclotide-bearing plant (Oldenlandia affinis) on nitrogen-free agar media supplemented with 15N salts. [Mylne & Craik, 2008].

The thia zip cyclization

The so-called thia zip reaction has proved very efficient for cyclization of disulfide-rich knottins. One efficient way to produce peptide cyclization is based on a transthioesterification between an N-terminal cysteine and a C-terminal thioester that forms an intermediate α-amino thiolactone that rearrange itself via an S,N-acyl isomerization into a C-to-N cyclic peptide. When there are internal thiols in the sequence between the N- and C-termini, there is a cascade of thiol-thiolactone exchanges, starting from the cysteine closest to the C-terminus, that eventually leads to the largest cyclic thiolactone where the irreversible S,N-acyl isomerization can take place. This entire process, which is called the thia zip cyclization (see scheme below), has been largely used to synthesize cyclic knottins [Tam & Lu, 1998; Tam & Yu, 1997; Daly et al, 1999].