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

Anne-Marie Lund Winther,Kristian Kølby Kristensen,Anni Kumari,Michael Ploug

doi:10.1016/j.jlr.2021.100149

Abstract

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.

Highlights

Building on our previous biophysical data showing that bovine LPL (bLPL) has a Tm of only 34.8◦C [23], we chose Drosophila Schneider 2 (S2) cells as the heterologous host cells for human LPL (hLPL) expression because these cells grow efficiently at 25◦C compared with the 37◦C required for most mammalian expression systems
We obtained 0.3 mg/l cultured medium for hLPLR297A alone, 0.7 mg/l cultured medium for hLPLR297A coexpressed with hGPIHBP1, 2.2 mg/l cultured medium for hLPLR297A coexpressed with hLMF1, whereas a synergistic effect was obtained for hLPLR297A coexpressed with both hLMF1 and hGPIHBP1 (13.5 mg/l cultured medium)
After extensive washing with ∼25 ml buffer consisting of 0.9 M NaCl, 10 mM Bis-Tris, and 10% glycerol, the bound LPL was eluted with a 12 ml linear gradient from 0.9 to 2 M NaCl, and the elution of hLPL peaked at a conductivity of 78 mS/cm corresponding to 1.3 M NaCl (Fig. 2A)

Summary

Objectives

The primary objective of this study was to develop an efficient and robust protocol for the expression and purification of recombinant hLPL that could supply large quantities of high-quality LPL in a pure and stable form and with high specific enzymatic activity

Methods

Results

Conclusion