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



Protease inhibitors




Chemical Synthesis

Since the very first solid-phase syntheses of the knottins CMTI-III and EETI-II reported in the late 80s [Kupryszewski et al, 1986; Le-Nguyen et al, 1989], great advances have been achieved in terms of strategies and methodological techniques [Boulegue et al, 2006], e.g.

    Stepwise regioselective cysteine pairing
    Induction of disulfide connectivities with selenocysteine
    Use of appropriate solid support
It is worth noting that the last oxidative folding step yielding the native disulfide connectivity and the three-dimensional structure, usually involved air oxidation. To favor the correct disulfide pattern, it is sometimes necessary to allow for disulfide shuffling under mildly alkaline conditions through addition of combinations of reduced and oxidized low-molecular-weight thiols.

Native Chemical Ligation

The solid phase synthesis works well for small peptides (~30-40 residues). To synthesize longer peptides, the chemical ligation of pre-purified fragments is more efficient. During the past ten years, native chemical ligation (NCL), first introduced by Dawson et al. [Dawson et al, 1994] has been widely used for the synthesis of moderate-size proteins in general, and knottins in particular [Tam & Yu, 1997; Daly et al, 1999; Buczek et al, 2005; Chino et al, 2006]. NCL consists in assembling an unprotected synthetic peptide-thioester with another unprotected segment containing an N-terminal cysteine residue to form a native peptide bond.

Initially designed for Boc-chemistry, NCL has been developped successfully for application in Fmoc-based peptide synthesis [Camarero & Mitchell, 2005], including numerous alternatives such as: