Solubility engineering and crystallization of human apolipoprotein D.
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 A, b = 75.1 A, c = 166.0 A, alpha = beta = gamma = 90 degrees), thus demonstrating its conformationally homogeneous behavior and providing a basis for the future X-ray structural analysis of this functionally still puzzling protein.
Nasreen
Amber
A
Lehrstuhl für Biologische Chemie, Technische Universität München, D-85350 Freising-Weihenstephan, Germany.
Vogt
Martin
M
Kim
Hyun Jin
HJ
Eichinger
Andreas
A
Skerra
Arne
A
D001053:Apolipoproteins / Q000737:chemistry* / Q000235:genetics*; D053399:Apolipoproteins D; D002850:Chromatography, Gel; D002942:Circular Dichroism; D003001:Cloning, Molecular / Q000379:methods; D003460:Crystallization; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D006801:Humans; D016297:Mutagenesis, Site-Directed; D015202:Protein Engineering / Q000379:methods; D012995:Solubility
D001053:Apolipoproteins; D053399:Apolipoproteins D