The KNOTTIN database

Chemical synthesis and 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:

• By extraction from natural sources [Favel et al, 1989; Saether et al, 1995; Hernandez et al, 2000; Cemazar et al, 2006]
• By chemical synthesis
• As expressed recombinant proteins [Camarero et al, 2007; Krause et al, 2007; Cui et al, 2007; Kimura et al, 2006; Kimura & Camarero, 2005; Ji et al, 2005; Schmoldt et al, 2005; Kratzner et al, 2005; Escoubas et al, 2003; Chen et al, 1992]

Synthesis	Expression	Protease inhibitors	Cyclotides	Toxins
Others

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],