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Solubility engineering and crystallization of human apolipoprotein D

Lehrstuhl für Biologische Chemie, Technische Universität München, D-85350 Freising-Weihenstephan, Germany

(RECEIVED August 15, 2005; FINAL REVISION September 30, 2005; ACCEPTED October 3, 2005)

Human apolipoprotein D (ApoD) is a physiologically important member of the lipocalin protein family that was discovered as a peripheral subunit of the high-density lipoprotein (HDL) but is also abundant in other body fluids and organs, including neuronal tissue. Although it has been possible to produce functional ApoD in the periplasm of Escherichia coli and to demonstrate its ligand-binding activity for progesterone and arachidonic acid, the recombinant protein suffers from a pronounced tendency to aggregate and to adsorb to vessel surfaces as well as chromatography matrices, thus hampering further structural investigation. Here, we describe a systematic mutagenesis study directed at presumably exposed hydrophobic side chains of the unglycosylated recombinant protein. As a result, one ApoD mutant with just three new amino acid substitutions—W99H, I118S, and L120S—was identified, which exhibits the following features: (1) improved yield upon periplasmic biosynthesis in E. coli, (2) elution as a monomeric protein from a gel permeation chromatography column, and (3) unchanged binding activity for its physiological ligands. In addition, the engineered ApoD was successfully crystallized (space group I4 with unit cell parameters a = 75.1 Å, b = 75.1 Å, c = 166.0 Å, = = = 90°), thus demonstrating its conformationally homogeneous behavior and providing a basis for the future X-ray structural analysis of this functionally still puzzling protein.

Keywords: E. coli expression; lipocalin; protein engineering; gel permeation chromatography; protein structure/folding; stability and mutagenesis; lipoproteins; isolation; characterization

Abbreviations: ApoD, apolipoprotein D • BBP, bilin-binding protein • GPC, gel permeation chromatography • HDL, high-density lipoprotein • IMAC, immobilized metal affinity chromatography • K_D, dissociation constant • OmpA, outer membrane protein A

Article published online ahead of print. Article and publication date are at http://www.proteinscience.org/cgi/doi/10.1110/ps.051775606.

Reprint requests to: Arne Skerra, Lehrstuhl für Biologische Chemie, Technische Universität München, D-85350 Freising-Weihenstephan, Germany; e-mail: skerra{at}wzw.tum.de; fax: +49-8161-714352.

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