Human Low Density Lipoprotein Receptor Fragment
SUCCESSFUL REFOLDING OF A FUNCTIONALLY ACTIVE LIGAND-BINDING DOMAIN PRODUCED IN ESCHERICHIA COLI*
- From the Gladstone Institute of Cardiovascular Disease, Cardiovascular Research Institute, Department of Pathology, University of California, San Francisco, California 94110
Abstract
The low density lipoprotein (LDL) receptor plays a key role in cholesterol homeostasis, mediating cellular uptake of lipoprotein particles by high affinity binding to its ligands, apolipoprotein (apo) B-100 and apoE. The ligand-binding domain of the LDL receptor contains 7 cysteine-rich repeats of approximately 40 amino acids; each repeat contains 6 cysteines, which form 3 intra-repeat disulfide bonds. As a first step toward determining the structure of the LDL receptor, both free and bound to its ligands, we produced inEscherichia coli a soluble fragment containing the ligand-binding domain (residues 1–292) as a thrombin-cleavable, heat-stable thioredoxin fusion. Modest amounts (5 mg/liter) of partially purified but inactive fragment were obtained after cell lysis, heat treatment, thrombin cleavage, and gel filtration under denaturing conditions. We were able to refold the receptor fragment to an active conformation with approximately 10% efficiency. The active fragment was isolated and purified with an LDL affinity column. The refolded receptor fragment was homogeneous, as determined by sodium dodecyl sulfate or non-denaturing polyacrylamide gel electrophoresis and isoelectric focusing. The purified fragment did not react with fluorescein-5-maleimide, indicating that all 42 cysteines were disulfide linked. In addition, the refolded fragment exhibited properties identical to those of the intact native receptor: Ca2+-dependent binding and isoform-dependent apoE binding (apoE2 binding <5% of apoE3). Furthermore, antibodies to the fragment recognized native receptors and inhibited the binding of 125I-LDL to fibroblast LDL receptors. We conclude that we have produced a properly folded and fully active receptor fragment that can be used for further structural studies.
Footnotes
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↵* This work was supported by the NIH Program Project Grant HL41633. The work described was carried out in part at the General Clinical Research Ctr., San Francisco General Hospital Medical Center with support from the National Center for Research Resources, NIH Grant RR00083.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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↵‡ To whom correspondence should be addressed: Gladstone Institute of Cardiovascular Disease, P.O. Box 419100, San Francisco, CA 94141-9100. Tel.: 415-826-7500; Fax: 415-285-5632; E-mail:karl_weisgraber.GICD{at}quickmail.ucsf.edu.
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↵1 The abbreviations used are: LDL, low density lipoprotein; apo, apolipoprotein; PAGE, polyacrylamide gel electrophoresis; DMPC, dimyristoyl phosphatidylcholine.
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- Received June 11, 1997.
- Revision received August 7, 1997.
