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 β2-adrenergic receptor (rhβ2AR) 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 rhβ2AR 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.
Highlights
The SMALP technology enables a direct solubilization of GPCRs from cell membranes.
The isolation of GPCRs from mammalian cells is usually a low-yield procedure.
SMALPs embedding the β2-adrenergic receptor were prepared from a HEK293T cell line.
The ionic strength of buffers plays a key role in SMALP formation and isolation.
Standard SMALP protocols can be finely optimized to increase purification yields.
Competing Interest Statement
The authors have declared no competing interest.
Abbreviations
- 2D-LC
- two-dimensional liquid chromatography
- AIBN
- 2,2’-azobisisobutyronitrile
- ATR-FTIR
- attenuated total reflectance Fourier-transform infrared
- DLS
- dynamic light scattering
- DMEM
- Dulbecco’s Modified Eagle Medium
- EB
- elution buffer
- ECL
- enhanced chemiluminescence
- FBS
- fetal bovine serum
- GPCR
- G protein-coupled receptor
- HPLC
- high-performance liquid chromatography
- HRP
- horseradish peroxidase
- IMAC
- immobilized metal affinity chromatography
- KP
- potassium phosphate buffer
- MAc
- maleic acid
- MAn
- maleic anhydride
- MP
- membrane protein
- Ni–NTA
- nickel(II)–nitrilotriacetic acid
- NMR
- nuclear magnetic resonance
- PCR
- polymerase chain reaction
- PTM
- post-translational modification
- RB
- running buffer
- rhβ2AR
- recombinant human β2-adrenergic receptor
- SB
- suspension buffer
- SEC
- size-exclusion chromatography
- SMAc
- styrene–maleic acid co-polymer
- SMAn
- styrene–maleic anhydride co-polymer
- SMALP
- styrene–maleic acid lipid particle
- ssHA
- hemagglutinin signal sequence
- Sty
- styrene
- TEV
- tobacco etch virus
- THF
- tetrahydrofuran