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




Serine protease inhibitors

Combined chemical and recombinant route to cyclic squash inhibitor analogs

In this approach, a cheap and high-yelding recombinant production of folded and oxidized linear presursors is combined with efficient chemical linkage of the termini to yield the cyclic knottins [Avrutina et al, 2008].

Biomimetic synthesis of the cyclic squash inhibitor MCoTI-II

In 2008, Thongyoo et al. [Thongyoo et al, 2008] proposed a new biomimetic method for the synthesis of MCoTI-II and analogs. In this method, a commercially available polymer-supported protease (trypsin or chymotrypsin) acts as a ligase for cyclisation of the refolded MCoTI-II backbone. The method also allows in situ affinity purification of the cyclic knottin.

Biosynthesis of the cyclic squash inhibitor MCoTI-II

A fully functional sample of he cyclic knottin MCoTI-II has been produced in living bacterial cells via an intramolecular version of the native chemical ligation using intein. [Camarero et al, 2007].

Synthesis of a photoactive analog of the squash inhibitor EETI-II

Using a combination of solid phase-peptide synthesis and chemical ligation, a photoactive methionine was incorporated into EETI-II. This or similar molecules will be useful for photoaffinity cross-linking studies [Durek et al, 2007].

Use of a Super Permeable Organic Combinatirial Chemistry (SPOCC) resin

In 2006, Johnson et al. [Johnson et al, 2006] reported an original synthesis of EETI-II based on the use of a superpermeable organic combinatorial chemistry (SPOCC) resin and thiazolidine-4-carboxylic acid (Thz) in place of cysteine. The combination of SPOCC resin and Thz allowed all synthetic steps to be achieved directly on the solid support, including 3 NCL and folding.

Early syntheses of CMTI-III and EETI-II

The squash inhibitors were among the first knottins to be chemically synthesized in the late 80s [Kupryszewski et al, 1986; Le-Nguyen et al, 1989],