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bioRxiv · 10.1101/2021.01.26.428305

Application of the SMALP technology to the isolation of GPCRs from low-yielding cell lines

Tedesco, D.·Maj, M.·Malarczyk, P.·Cingolani, A.·Zaffagnini, M.·Wnorowski, A.·Benelli, T.·Mazzoni, R.·Bartolini, M.·Jozwiak, K.
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Abstract

The ability of styrene-maleic acid (SMAc) co-polymers to spontaneously insert into biological membranes can be exploited to extract G protein-coupled receptors (GPCRs) embedded in styrene-maleic acid lipid particles (SMALPs), preserving the native environment around the protein and thus enhancing the feasibility of functional studies. So far, the SMALP technology has been primarily employed on non-mammalian cells and protocols are not optimized for adherent human cell lines, which cannot be harvested in large amounts. In this work, a fine investigation of key parameters affecting the formation of SMALPs was undertaken with the purpose of maximizing the yield of extraction of a recombinant form of human {beta}2-adrenergic receptor (rh{beta}2AR) from HEK293T cells. The study highlighted an important influence of ionic strength on the membrane solubilization efficiency and GPCR purification yield of SMAc co-polymers: by lowering the salt concentration of all buffers used in previously published SMALP protocols, the water solubility and extraction efficiency of the selected SMAc co-polymer (commercially supplied as a potassium salt) were enhanced. In-line combination of size-exclusion chromatography (SEC) with immobilized metal affinity chromatography (IMAC) allowed further improvement of the final rh{beta}2AR yield by reducing the loss of SMALP-embedded GPCRs during the fractionation and purification of SMALPs. The overall findings of this study show that the available SMALP protocols can be significantly optimized in several aspects in order to increase the efficiency of GPCR solubilization and isolation from low-yielding expression systems. HighlightsO_LIThe SMALP technology enables a direct solubilization of GPCRs from cell membranes. C_LIO_LIThe isolation of GPCRs from mammalian cells is usually a low-yield procedure. C_LIO_LISMALPs embedding the {beta}2-adrenergic receptor were prepared from a HEK293T cell line. C_LIO_LIThe ionic strength of buffers plays a key role in SMALP formation and isolation. C_LIO_LIStandard SMALP protocols can be finely optimized to increase purification yields. C_LI Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=57 SRC="FIGDIR/small/428305v1_ufig1.gif" ALT="Figure 1"> View larger version (34K): org.highwire.dtl.DTLVardef@14a3bdcorg.highwire.dtl.DTLVardef@a00d14org.highwire.dtl.DTLVardef@1baca87org.highwire.dtl.DTLVardef@4686c7_HPS_FORMAT_FIGEXP M_FIG C_FIG

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Tedesco, D., Maj, M., Malarczyk, P., Cingolani, A., Zaffagnini, M., Wnorowski, A., Benelli, T., Mazzoni, R., Bartolini, M., Jozwiak, K.. 2021-01-27. Application of the SMALP technology to the isolation of GPCRs from low-yielding cell lines. https://doi.org/10.1101/2021.01.26.428305

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