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G-protein coupled receptor solubilization and purification for biophysical analysis and functional studies, in the total absence of detergent.

Jamshad M, Charlton J, Lin YP, Routledge SJ, Bawa Z, Knowles TJ, Overduin M, Dekker N, Dafforn TR, Bill RM, Poyner DR, Wheatley M - Biosci. Rep. (2015)

Bottom Line: Furthermore, the A2AR-SMALP, generated from yeast (Pichia pastoris) or mammalian cells, exhibited increased thermostability (~5°C) compared with detergent [DDM (n-dodecyl-β-D-maltopyranoside)]-solubilized A2AR controls.Moreover, in contrast with nanodiscs stabilized by scaffold proteins, the non-proteinaceous nature of the SMA polymer allowed unobscured biophysical characterization of the embedded receptor.Consequently, CD spectroscopy was used to relate changes in secondary structure to loss of ligand binding ([(3)H]ZM241385) capability.

View Article: PubMed Central - PubMed

Affiliation: *School of Biosciences, University of Birmingham, Edgbaston, Birmingham, B15 2TT, U.K.

ABSTRACT
G-protein coupled receptors (GPCRs) constitute the largest class of membrane proteins and are a major drug target. A serious obstacle to studying GPCR structure/function characteristics is the requirement to extract the receptors from their native environment in the plasma membrane, coupled with the inherent instability of GPCRs in the detergents required for their solubilization. In the present study, we report the first solubilization and purification of a functional GPCR [human adenosine A2A receptor (A2AR)], in the total absence of detergent at any stage, by exploiting spontaneous encapsulation by styrene maleic acid (SMA) co-polymer direct from the membrane into a nanoscale SMA lipid particle (SMALP). Furthermore, the A2AR-SMALP, generated from yeast (Pichia pastoris) or mammalian cells, exhibited increased thermostability (~5°C) compared with detergent [DDM (n-dodecyl-β-D-maltopyranoside)]-solubilized A2AR controls. The A2AR-SMALP was also stable when stored for prolonged periods at 4°C and was resistant to multiple freeze-thaw cycles, in marked contrast with the detergent-solubilized receptor. These properties establish the potential for using GPCR-SMALP in receptor-based drug discovery assays. Moreover, in contrast with nanodiscs stabilized by scaffold proteins, the non-proteinaceous nature of the SMA polymer allowed unobscured biophysical characterization of the embedded receptor. Consequently, CD spectroscopy was used to relate changes in secondary structure to loss of ligand binding ([(3)H]ZM241385) capability. SMALP-solubilization of GPCRs, retaining the annular lipid environment, will enable a wide range of therapeutic targets to be prepared in native-like state to aid drug discovery and understanding of GPCR molecular mechanisms.

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Related in: MedlinePlus

Purification of SMALP-solubilized His-tagged A2AR from P. pastoris(a) The A2AR eluted from the Ni2+–NTA linked agarose as a single band in silver-stained fractions with 250 mM imidazole. (b) Western blot of the 250 mM imidazole fraction with an anti-histidine antibody.
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Figure 1: Purification of SMALP-solubilized His-tagged A2AR from P. pastoris(a) The A2AR eluted from the Ni2+–NTA linked agarose as a single band in silver-stained fractions with 250 mM imidazole. (b) Western blot of the 250 mM imidazole fraction with an anti-histidine antibody.

Mentions: The yeast P. pastoris has been utilized extensively to overexpress a wide range of membrane proteins from a variety of organisms, including GPCRs for crystallization [24,25]. A histidine-tagged human A2AR was expressed in a genetically-engineered strain of P. pastoris, developed specifically for high-level expression of recombinant membrane protein [26], using culture conditions optimized for production of functional receptor. A2AR-expressing yeast cells were disrupted [16], then directly solubilized with 2.5% (w/v) SMA co-polymer in the total absence of detergent. The membrane pellet visibly clarified upon exposure to SMA due to SMALP formation. Following removal of non-solubilized material by centrifugation (100000 g, 1h), the A2AR–SMALP was purified using Ni2+–NTA linked agarose (Figure 1a). The A2AR eluted as a single band in fractions containing 250 mM imidazole. The identity of the band as the A2AR was confirmed by Western blotting using anti-histidine antibody to detect the histidine-tagged A2AR (Figure 1b) and partial sequencing of the purified protein using Fourier-transform ion cyclotron resonance (FTICR) MS. The FTICR identified three peptides YNGLVTGTR, QMESQPLPGER and SHVLRQQEPFK corresponding to A2AR residues 112–120 (part of intracellular loop 2), 210–220 (part of intracellular loop 3) and 305–315 (part of the C-terminal tail) respectively.


G-protein coupled receptor solubilization and purification for biophysical analysis and functional studies, in the total absence of detergent.

Jamshad M, Charlton J, Lin YP, Routledge SJ, Bawa Z, Knowles TJ, Overduin M, Dekker N, Dafforn TR, Bill RM, Poyner DR, Wheatley M - Biosci. Rep. (2015)

Purification of SMALP-solubilized His-tagged A2AR from P. pastoris(a) The A2AR eluted from the Ni2+–NTA linked agarose as a single band in silver-stained fractions with 250 mM imidazole. (b) Western blot of the 250 mM imidazole fraction with an anti-histidine antibody.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4400634&req=5

Figure 1: Purification of SMALP-solubilized His-tagged A2AR from P. pastoris(a) The A2AR eluted from the Ni2+–NTA linked agarose as a single band in silver-stained fractions with 250 mM imidazole. (b) Western blot of the 250 mM imidazole fraction with an anti-histidine antibody.
Mentions: The yeast P. pastoris has been utilized extensively to overexpress a wide range of membrane proteins from a variety of organisms, including GPCRs for crystallization [24,25]. A histidine-tagged human A2AR was expressed in a genetically-engineered strain of P. pastoris, developed specifically for high-level expression of recombinant membrane protein [26], using culture conditions optimized for production of functional receptor. A2AR-expressing yeast cells were disrupted [16], then directly solubilized with 2.5% (w/v) SMA co-polymer in the total absence of detergent. The membrane pellet visibly clarified upon exposure to SMA due to SMALP formation. Following removal of non-solubilized material by centrifugation (100000 g, 1h), the A2AR–SMALP was purified using Ni2+–NTA linked agarose (Figure 1a). The A2AR eluted as a single band in fractions containing 250 mM imidazole. The identity of the band as the A2AR was confirmed by Western blotting using anti-histidine antibody to detect the histidine-tagged A2AR (Figure 1b) and partial sequencing of the purified protein using Fourier-transform ion cyclotron resonance (FTICR) MS. The FTICR identified three peptides YNGLVTGTR, QMESQPLPGER and SHVLRQQEPFK corresponding to A2AR residues 112–120 (part of intracellular loop 2), 210–220 (part of intracellular loop 3) and 305–315 (part of the C-terminal tail) respectively.

Bottom Line: Furthermore, the A2AR-SMALP, generated from yeast (Pichia pastoris) or mammalian cells, exhibited increased thermostability (~5°C) compared with detergent [DDM (n-dodecyl-β-D-maltopyranoside)]-solubilized A2AR controls.Moreover, in contrast with nanodiscs stabilized by scaffold proteins, the non-proteinaceous nature of the SMA polymer allowed unobscured biophysical characterization of the embedded receptor.Consequently, CD spectroscopy was used to relate changes in secondary structure to loss of ligand binding ([(3)H]ZM241385) capability.

View Article: PubMed Central - PubMed

Affiliation: *School of Biosciences, University of Birmingham, Edgbaston, Birmingham, B15 2TT, U.K.

ABSTRACT
G-protein coupled receptors (GPCRs) constitute the largest class of membrane proteins and are a major drug target. A serious obstacle to studying GPCR structure/function characteristics is the requirement to extract the receptors from their native environment in the plasma membrane, coupled with the inherent instability of GPCRs in the detergents required for their solubilization. In the present study, we report the first solubilization and purification of a functional GPCR [human adenosine A2A receptor (A2AR)], in the total absence of detergent at any stage, by exploiting spontaneous encapsulation by styrene maleic acid (SMA) co-polymer direct from the membrane into a nanoscale SMA lipid particle (SMALP). Furthermore, the A2AR-SMALP, generated from yeast (Pichia pastoris) or mammalian cells, exhibited increased thermostability (~5°C) compared with detergent [DDM (n-dodecyl-β-D-maltopyranoside)]-solubilized A2AR controls. The A2AR-SMALP was also stable when stored for prolonged periods at 4°C and was resistant to multiple freeze-thaw cycles, in marked contrast with the detergent-solubilized receptor. These properties establish the potential for using GPCR-SMALP in receptor-based drug discovery assays. Moreover, in contrast with nanodiscs stabilized by scaffold proteins, the non-proteinaceous nature of the SMA polymer allowed unobscured biophysical characterization of the embedded receptor. Consequently, CD spectroscopy was used to relate changes in secondary structure to loss of ligand binding ([(3)H]ZM241385) capability. SMALP-solubilization of GPCRs, retaining the annular lipid environment, will enable a wide range of therapeutic targets to be prepared in native-like state to aid drug discovery and understanding of GPCR molecular mechanisms.

Show MeSH
Related in: MedlinePlus