DARPins

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Structural model of a DARPin based on X-ray structural analysis data (PDB 2QYJ)

DARPins (short from Designed Ankyrin Repeat Proteins ) are artificial proteins that are capable of recognizing and binding antigens . They are structurally derived from ankyrin proteins and consist of several repeat motifs of these proteins. DARPins with a total of four repeat motifs and a molecular mass of about 14  kDa are about 10 times lighter than an antibody of the IgG type. Their affinity for an antigen is comparable to that of an antibody. DARPins can act as agonists , antagonists , inverse agonists, or enzyme inhibitors . DARPins were largely developed at the University of Zurich . The aim is to use them as tools in research as well as diagnostics and therapeutics .

structure

DARPins are structurally derived from proteins that carry one or more so-called ankyrin repeat motifs. This widespread motif , usually 33 amino acid long, consists of a β-loop and two α-helices and can be found not only in the cytoskeletal protein ankyrin, but also in large numbers in proteins of almost all living things. DARPins have at least three of these motifs. Even smaller artificial repeat proteins with only one or two repeat motifs do not form a sufficient tertiary structure . DARPins with two or three variable repeat motifs, which are flanked by two terminal constant repeat motifs, are developed and used in particular as antibody mimetics .

The targeted exchange of individual amino acids within the repeat motif can contribute to an increase in affinity. The amino acids close to the surface in positions 1, 3, 11, 12, 31 and 33 of the respective motifs are suitable for modifications, since they are not essential for the structure of the DARPins, but are associated with an interaction with target proteins.

properties

Like other antibody mimetics, DARPins have a higher temperature stability than most antibodies. A denaturation of DARPins with four to six repeat motifs takes place only at temperatures above the range 66-85 ° C.

DARPins, which are smuggled into target cells using genetic engineering methods, are suitable as alternative tools to intrabodies and RNAi for studying cell functions and represent an option in gene therapy .

Manufacturing

With the help of molecular biological methods by applying the random mutagenesis are molecular libraries created by DARPins. These DARPin libraries are selected using suitable display techniques, such as phage display or ribosome display , DARPins which can bind the target protein. Once the most suitable DARPin has been selected, it can be produced with a very high yield with the help of production organisms such as Escherichia coli .

Individual evidence

  1. ↑ Stumpp MT, Binz HK, Amstutz P: DARPins: a new generation of protein therapeutics . In: Drug Discov Today . 13, No. 15-16, August 2008, pp. 695-701. doi : 10.1016 / j.drudis.2008.04.013 . PMID 18621567 .
  2. ↑ Heinzelmann E (2008). DARPins - all-rounder for diagnosis and therapy. ( Memento of March 5, 2012 in the Internet Archive ) (PDF; 142 kB) Swiss Engineering 12/2008: 62-63.
  3. ↑ Bork P: Hundreds of ankyrin-like repeats in functionally diverse proteins: mobile modules that cross phyla horizontally? . In: Proteins . 17, No. 4, December 1993, pp. 363-74. doi : 10.1002 / prot.340170405 . PMID 8108379 .
  4. ↑ Mosavi LK, Minor DL, Peng ZY: Consensus-derived structural determinants of the ankyrin repeat motif . In: Proc. Natl. Acad. Sci. USA . 99, No. 25, December 2002, pp. 16029-34. doi : 10.1073 / pnas.252537899 . PMID 12461176 . PMC 138559 (free full text).
  5. ↑ Stumpp MT, Amstutz P: DARPins: a true alternative to antibodies . In: Curr Opin Drug Discov Dev . 10, No. 2, March 2007, pp. 153-9. PMID 17436550 .
  6. ↑ a b Binz HK, Stumpp MT, Forrer P, Amstutz P, Plückthun A: Designing repeat proteins: well-expressed, soluble and stable proteins from combinatorial libraries of consensus ankyrin repeat proteins . In: J. Mol. Biol. . 332, No. 2, September 2003, pp. 489-503. PMID 12948497 .
  7. ↑ Amstutz P, Binz HK, Parizek P, et al. : Intracellular kinase inhibitors selected from combinatorial libraries of designed ankyrin repeat proteins . In: J Biol Chem . 280, No. 26, July 2005, pp. 24715-22. doi : 10.1074 / jbc.M501746200 . PMID 15851475 .

literature