Construction of an artificial receptor protein ("anticalin") based on the human apolipoprotein D.
Human apolipoprotein D (ApoD) is a prominent member of the lipocalin family of proteins and transports arachidonic acid and progesterone in various body fluids. Lipocalins share a structurally conserved beta barrel as their central folding unit, which supports a set of four hypervariable loops that form the entrance to the ligand pocket. Based on this structural pattern ApoD was employed as a scaffold for the combinatorial design of artificial receptor proteins termed anticalins. After randomization of 24 amino acids located within the loop region, several ApoD variants were selected against hemoglobin, a biochemically well-characterized model target, by using bacterial phagemid display and colony screening. One variant, dubbed HbgA, was further investigated by surface plasmon resonance interaction analysis and found to complex hemoglobin specifically and with a dissociation constant of about 2 microM. While our previous work on the structurally related insect bilin-binding protein was focused on the generation of binding activity towards low-molecular-weight ligands, this study demonstrates for the first time that a lipocalin can also be tailored to recognize a protein target. The fact that even a human member of this protein family has now been successfully recruited for anticalin construction opens the possibility for future application of such engineered lipocalins as target-recognition vehicles in medical therapy.
Vogt
Martin
M
Lehrstuhl für Biologische Chemie, Technische Universität München, 85350 Freising-Weihenstephan, Germany.
Skerra
Arne
A
D000595:Amino Acid Sequence; D001053:Apolipoproteins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D053399:Apolipoproteins D; D001483:Base Sequence; D002942:Circular Dichroism; D020650:Combinatorial Chemistry Techniques; D006454:Hemoglobins / Q000378:metabolism; D006801:Humans; D008024:Ligands; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D019151:Peptide Library; D011485:Protein Binding; D015202:Protein Engineering; D012995:Solubility
D001053:Apolipoproteins; D053399:Apolipoproteins D; D006454:Hemoglobins; D008024:Ligands; D019151:Peptide Library