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Expression and one-step purification of active LPL contemplated by biophysical considerations

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DOI

  1. ANGPTL4 sensitizes lipoprotein lipase to PCSK3 cleavage by catalyzing its unfolding

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  2. Chylomicronemia from GPIHBP1 autoantibodies

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  3. ANGPTL4 inactivates lipoprotein lipase by catalyzing the irreversible unfolding of LPL's hydrolase domain

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  4. The structural basis for monoclonal antibody 5D2 binding to the tryptophan-rich loop of lipoprotein lipase

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  5. On the mechanism of angiopoietin-like protein 8 for control of lipoprotein lipase activity

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  1. ANGPTL4: a new mode in the regulation of intravascular lipolysis

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  2. The Urokinase Receptor (uPAR) as a "Trojan Horse" in Targeted Cancer Therapy: Challenges and Opportunities

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  3. The Importance of Lipoprotein Lipase Regulation in Atherosclerosis

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  4. ANGPTL4 sensitizes lipoprotein lipase to PCSK3 cleavage by catalyzing its unfolding

    Publikation: Bidrag til tidsskriftTidsskriftartikelForskningpeer review

Vis graf over relationer

LPL is essential for intravascular lipid metabolism and is of high medical relevance. Since LPL is notoriously unstable, there is an unmet need for a robust expression system producing high quantities of active and pure recombinant human LPL (hLPL). We showed previously that bovine LPL purified from milk is unstable at body temperature (Tm is 34.8°C), but in the presence of the endothelial transporter glycosylphosphatidylinositol-anchored high density lipoprotein-binding protein 1 (GPIHBP1), LPL is stabile (Tm increases to 57.6°C). Building on this information, we now designed an expression system for hLPL using Drosophila Schneider 2 cells grown in suspension at high cell density and at an advantageous temperature of 25°C. We cotransfected Schneider 2 cells with hLPL, lipase maturation factor 1, and soluble GPIHBP1 to provide an efficient chaperoning and stabilization of LPL in all compartments during synthesis and after secretion into the conditioned medium. For LPL purification, we used heparin-Sepharose affinity chromatography, which disrupted LPL-GPIHBP1 complexes causing GPIHBP1 to elute with the flow-through of the conditioned media. This one-step purification procedure yielded high quantities of pure and active LPL (4-28 mg/l). Purification of several hLPL variants (furin cleavage-resistant mutant R297A, active-site mutant S132A, and lipid-binding-deficient mutant W390A-W393A-W394A) as well as murine LPL underscores the versatility and robustness of this protocol. Notably, we were able to produce and purify LPL containing the cognate furin cleavage site. This method provides an efficient and cost-effective approach to produce large quantities of LPL for biophysical and large-scale drug discovery studies.

OriginalsprogEngelsk
Artikelnummer100149
TidsskriftJournal of Lipid Research
Vol/bind62
Sider (fra-til)100149
ISSN0022-2275
DOI
StatusUdgivet - 12 nov. 2021

Bibliografisk note

Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.

ID: 69168762