• • 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]

Recombinant protein expression

Since knottins are essentially very small proteins, chemical synthesis has long been the method of choice to produce these miniproteins in large quantities. The use of bacterial or eukariotic expression systems can be however a cost-effective alternative approach for production of knottins without posttranslational modifications. It also opens the route toward large combinatorial libraries by using the classical tools of molecular biology.

Expression systems

• Due to low cost and short culturing times, Escherichia coli is certainly the most used expression system. To avoid quick degradation, improve the expression level, facilitate purification and/or obtain soluble expressed proteins, disulfide-rich miniproteins were often produced in fusion with larger proteins e.g. maltose-binding protein, thioredoxin, glutathione S-transferase or Barnase [Becker & Terlau 2008].
• In contrast to Escherichia coli, eukariotic expression systems allow intracellular folding and expression into the surrounding medium and are well fitted to the production of dsulfide-rich proteins. Knottins were thus succesfully produced in yeast Pichia pastoris or in insect cells with the Baculovirus system.

Production of cyclic proteins

Some knottins, mostly cyclotides, are cyclic with a circular backbone in which the C-terminus is linked to the N-terminus via a regular peptide bond. This cyclization post-tranlational event, which is supposed to occur through the involvement of an arginyl endopeptidase in vivo, is a challenge for recombinant expression systems. One cyclic knottin has been produced in living bacterial cells via an intramolecular version of the native chemical ligation using intein. [Camarero et al, 2007].
In another 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 knottin. [Avrutina et al, 2008].