DARPin

DARPins ( shortly. Designed ankyrin repeat protein of engl ) are artificial proteins that are capable of recognizing and binding of 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. Its affinity for an antigen similar to that of an antibody. DAPRins can behave as agonists, antagonists, inverse agonists or enzyme inhibitors. DARPins were largely developed at the University of Zurich. A use as tools in research as well as diagnostic and therapeutic agents is sought.

Structure

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

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

Properties

As other antibody mimics possess a DARPins against most antibodies increased temperature stability. Denaturation of DARPins having four to six repeat motifs is effected at temperatures above the range 66-85 ° C.

DARPins that are introduced by genetic engineering methods in target cells, can be used as alternative tools to intra bodysuits and RNAi for the study of cell function and provide an option in gene therapy dar.

Production

Using molecular biological methods using random mutagenesis, molecular libraries of DARPins be created. DARPin these libraries can be selected using the appropriate display techniques, such as the phage display or ribosome display, DARPins that bind the target protein. After a selection of the most suitable DARPins can this with a very high yield with the help of producer organisms such as Escherichia coli produce.