Affilin-novel binding molecules based on human gamma-B-crystallin, an all beta-sheet protein.
The concept of novel binding proteins as an alternative to antibodies has undergone rapid development and is now ready for practical use in a wide range of applications. Alternative binding proteins, based on suitable scaffolds with desirable properties, are selected from combinatorial libraries in vitro. Here, we describe an approach using a beta-sheet of human gamma-B-crystallin to generate a universal binding site through randomization of eight solvent-exposed amino acid residues selected according to structural and sequence analyses. Specific variants, so-called Affilin, have been isolated from a phage display library against a variety of targets that differ considerably in size and structure. The isolated Affilin variants can be produced in Escherichia coli as soluble proteins and have a high level of thermodynamic stability. The crystal structures of the human wild-type gamma-B-crystallin and a selected Affilin variant have been determined to 1.7 A and 2.0 A resolution, respectively. Comparison of the two molecules indicates that the human gamma-B-crystallin tolerates amino acid exchanges with no major structural change. We conclude that the intrinsically stable and easily expressed gamma-B-crystallin provides a suitable framework for the generation of novel binding molecules.
Ebersbach
Hilmar
H
Scil Proteins GmbH, Heinrich Damerow Str. 1, 06120 Halle (Saale), Germany. hebersbach@bioc.unizh.ch
Fiedler
Erik
E
Scheuermann
Tanja
T
Fiedler
Markus
M
Stubbs
Milton T
MT
Reimann
Carola
C
Proetzel
Gabriele
G
Rudolph
Rainer
R
Fiedler
Ulrike
U
D000818:Animals; D002352:Carrier Proteins / Q000138:chemical synthesis* / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D002417:Cattle; D004958:Estradiol / Q000378:metabolism; D005240:Feasibility Studies; D006801:Humans; D007074:Immunoglobulin G / Q000378:metabolism; D008958:Models, Molecular; D020932:Nerve Growth Factor / Q000378:metabolism; D019151:Peptide Library; D011485:Protein Binding; D015202:Protein Engineering / Q000379:methods*; D011498:Protein Precursors / Q000378:metabolism; D017433:Protein Structure, Secondary; D013379:Substrate Specificity; D013739:Testosterone / Q000378:metabolism; D038222:gamma-Crystallins / Q000737:chemistry*
D002352:Carrier Proteins; D007074:Immunoglobulin G; D019151:Peptide Library; D011498:Protein Precursors; C107738:crystallin, gammaB; D038222:gamma-Crystallins; C061397:pro-nerve growth factor, human; D013739:Testosterone; D004958:Estradiol; D020932:Nerve Growth Factor