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].
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
- 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].