The great interest in the identification of protein-protein interactions culminated in the first selection system which was phage display in the middle of the eighties. Beginning of the nineties a method for the in vitro selection of nucleic acids was introduced, which enables the rapid generation of aptamers and ribozymes and highlighted the enormous advantages and possibilities of an in vitro system stemming from the large initial libraries with up to 1016 different members. Because proteins in contrast to nucleic acids carry out a wider range of structural and catalytic roles in biology and are much more extensively used in diagnostic, therapeutic, and industrial applications, great interest has been generated in the development of methods for the in vitro selection of proteins. At the end of the nineties a method for the in vitro selection and evolution of proteins was developed and designated ribosome display.
This work describes the selection of streptavidin-binding peptides and the development of the required technologies. Streptavidin was chosen as target molecule, because a large number of commercial available streptavidin-conjugates is widely used in the field of life science. A cell-free, prokaryotic translation system was the basis for establishing the ribosome display technique and took place with help of a model system. For the first time such a model system was based on a simple affinity tag, namely His-tag and not an antibody. Two nucleic acid encoded peptide libraries had been generated. The random portion of the one library consisted of 16 NNS-codons and encodes the loopstructure of the chymotrypsin inhibitor 2 from barley seeds. The other library contained 15 NNS-codons and encodes the N-terminus of the fatty acid binding protein (FABP) from bovine heart. It was possible to isolate streptavidin-binding peptides from the combinatorial FABP-library after seven rounds of in vitro selection. Cloning and sequencing revealed a number of different peptides which could be divided in two groups. The larger group involves peptides with a HPQ-motif which is known from other selections. The other group involves totally new peptides with a DVEAW-motif at their N-terminus. Out of this group the best binder evolves with the sequence DVEAWLDERV-PLVET and a Kd value of about 84 nM. This peptide is outstandingly suitable as an affinity peptide for the purification of recombinant proteins.
