ANTICALINS: Protein Engineering of Lipocalins for Novel Binding Functions
Arne Skerra

ANTICALINS: Protein Engineering of Lipocalins for Novel Binding Functions

Arne Skerra, PhD., Professor of Biological Chemistry, Technical University Munich, Germany

Lipocalins are a widespread family of small and robust proteins, which usually serve for the transport or storage of poorly soluble or chemically sensitive biological compounds, including vitamins, steroid hormones, odorants, and various secondary metabolites [1]. Despite extremely low mutual sequence homology they share a common tertiary structure, which is dominated by a circular eight-stranded anti-parallel b-sheet with an a-helix attached to it. At one end, this b-barrel supports four structurally variable loops, which form the entrance to the binding pocket. There are approximately ten different lipocalins in the human body, among which the plasma retinol-binding protein constitutes the most prominent member. We are currently active in the structural elucidation of several of these proteins via X-ray crystallography, for example human tear lipocalin [2].

Apart from studying their natural ligand-binding functions, we have employed lipocalins as scaffolds for the design of artificial binding proteins termed 'anticalins' [3]. In a combinatorial protein design approach we first set out to reshape the ligand pocket of the bilin-binding protein from Pieris brassicae. A library was prepared by subjecting 16 amino acid positions in the four loops of this prototypic lipocalin to simultaneous random mutagenesis, followed by panning with different immobilized compounds via phage display [4]. Thus, mutants with KD values in the low nanomolar range were selected against several hapten targets, including fluorescein and digoxigenin. Crystallographic analyses of these anticalins revealed high structural plasticity of the loop region and illustrated the pronounced specificity in the molecular recognition of the low molecular weight ligands.

Meanwhile, anticalins based on human lipocalins and with specificities for protein targets, especially disease-related cell surface receptors, were generated, as well. Compared with recombinant antibodies or their fragments, anticalins provide several practical benefits because they are composed of a single polypeptide chain, have a much smaller size, and their set of four loops can be easily manipulated at the genetic level. Anticalins can be produced at high amounts in E. coli and they exhibit surprising stability, with typical denaturation temperatures above 60 °C. In addition, they are amenable to the production of functional fusion proteins. Hence, specifically engineered anticalins offer advantageous properties for substituting conventional antibodies in several areas [5], including medical therapy [1].

References:
[1] Schlehuber, S. & Skerra, A. (2005) Lipocalins in drug discovery: from natural ligand-binding proteins to 'anticalins'. Drug Discov. Today 10, 23-33.
[2] Breustedt, D. A., Korndörfer, I. P., Redl, B. & Skerra, A. (2005) The 1.8-Å crystal structure of human tear lipocalin reveals an extended branched cavity with capacity for multiple ligands. J. Biol. Chem. 280, 484-493.
[3] Skerra, A. (2000) Lipocalins as a scaffold. Biochim. Biophys. Acta 1482, 337-350.
[4] Beste, G., Schmidt, F. S., Stibora, T. & Skerra, A. (1999) Small antibody-like proteins with prescribed ligand specificities derived from the lipocalin fold. Proc. Natl. Acad. Sci. USA 96, 1898-1903.
[5] Skerra, A. (2003) Imitating the humoral immune response. Curr. Opin. Chem. Biol. 7, 683-693.