Purification of the Cystic Fibrosis Transmembrane Conductance Regulator Protein Expressed in &lt;em&gt;Saccharomyces cerevisiae&lt;/em&gt;

Pollock, Naomi L.; Cant, Natasha; Rimington, Tracy L.; Ford, Robert C.

doi:10.3791/51447

Cited by 16 publications

(13 citation statements)

References 26 publications

(31 reference statements)

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“…Using these same assay conditions, we then varied [ATP] to determine the Vmax for purified, PKA-phosphorylated human CFTR (Figure 4). For three independently purified CFTR preparations, the Vmax value was 240 ± 60 nmol/min/mg, a rate several fold greater than previous values of 30 nmol/min/mg reported by our own laboratory [13] or 13-77 nmol/min/mg by others [26, 27, 29, 30]. …”

Section: Resultscontrasting

confidence: 52%

Specific stabilization of CFTR by phosphatidylserine

Hildebrandt

Khazanov

Kappes

et al. 2017

Biochimica et Biophysica Acta (BBA) - Biomembranes

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The Cystic Fibrosis Transmembrane Conductance Regulator (CFTR, ABCC7) is a plasma membrane chloride ion channel in the ABC transporter superfamily. CFTR is a key target for cystic fibrosis drug development, and its structural elucidation would advance those efforts. However, the limited in vivo and in vitro stability of the protein, particularly its nucleotide binding domains, has made structural studies challenging. Here we demonstrate that phosphatidylserine uniquely stimulates and thermally stabilizes the ATP hydrolysis function of purified human CFTR. Among several lipids tested, the greatest stabilization was observed with brain phosphatidylserine, which shifted the Tm for ATPase activity from 22.7 ± 0.8 °C to 35.0 ± 0.2 °C in wild-type CFTR, and from 26.6 ± 0.7 °C to 42.1 ± 0.2 °C in a more stable mutant CFTR having deleted regulatory insertion and S492P/A534P/I539T mutations. When ATPase activity was measured at 37 °C in the presence of brain phosphatidylserine, Vmax for wild-type CFTR was 240 ± 60 nmol/min/mg, a rate higher than previously reported and consistent with rates for other purified ABC transporters. The significant thermal stabilization of CFTR by phosphatidylserine may be advantageous in future structural and biophysical studies of CFTR.

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Section: Resultscontrasting

confidence: 52%

Specific stabilization of CFTR by phosphatidylserine

Hildebrandt

Khazanov

Kappes

et al. 2017

Biochimica et Biophysica Acta (BBA) - Biomembranes

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“…This confirmed earlier reports of channel activity and ATP binding, detected using UV cross-linking of azido-ATP, by CFTR purified from the same cell line in the anionic detergent lyso-phosphatidyl glycerol (LPG)-16 [33]. Similarly, hydrolysis of ATP has been detected in protein purified from yeast in LPG-14 and reconstituted into phospholipids and cholesterol, albeit at a lower rate than the CFTR purified from the same cells using dodecylmaltoside [37].…”

Section: Purification Of Cftrsupporting

confidence: 89%

“…For biochemical and structural analyses, which typically require large quantities of purified protein, it has been necessary to develop heterologous overexpression systems. Several cell lines have been used, including insect Sf9 cells [32,33] and yeast [34][35][36][37]. Expression in mammalian cell lines was initially thought to be impossible [31], but has now been achieved using transient expression in baby hamster kidney (BHK) cells [38] and even a stable expression system in the human embryonic kidney (HEK)-293 cell line [39].…”

Section: Purification Of Cftrmentioning

confidence: 99%

“…Following previous purifications of CFTR and the considerations described above, we have developed an expression system for CFTR in Saccharomyces cerevisiae and begun to purify and characterize several orthologues of the protein, including chicken, mouse, platypus, salmon and human [37,61,62] (Tracy L. Rimington, Natasha Cant, Naomi L. Pollock, Ina Urbatsch and Robert C. Ford, unpublished data). The orthologues under consideration (Figure 1) [63] were chosen for their diversity across the animal kingdom, as Sequences were taken from the Uniprot database (accession numbers P13569, Q9W750, P26361, A0M8U4 and Q07DZ6 respectively).…”

Section: Exploiting Evolutionary Diversity In Cftrmentioning

confidence: 99%

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Characterizing diverse orthologues of the cystic fibrosis transmembrane conductance regulator protein for structural studies

Pollock

Rimington

Ford

2015

Biochemical Society Transactions

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As an ion channel, the cystic fibrosis transmembrane conductance regulator (CFTR) protein occupies a unique niche within the ABC family. Orthologues of CFTR are extant throughout the animal kingdom from sharks to platypods to sheep, where the osmoregulatory function of the protein has been applied to differing lifestyles and diverse organ systems. In humans, loss-of-function mutations to CFTR cause the disease cystic fibrosis, which is a significant health burden in populations of white European descent. Orthologue screening has proved fruitful in the pursuit of high-resolution structural data for several membrane proteins, and we have applied some of the princples developed in previous studies to the expression and purification of CFTR. We have overexpressed this protein, along with evolutionarily diverse orthologues, in Saccharomyces cerevisiae and developed a purification to isolate it in quantities sufficient for structural and functional studies.

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“…Protein purification: Cell rupture and microsome preparation were described previously (35). Microsomes were diluted to 2.5 mg/ml prior to solubilisation in detergent-containing buffer (50 mM Tris pH 8.0, 10% (v/v) glycerol, 50 mM NaCl, 1 mM 2-mercaptoethanol and 2% (w/v) DDM).…”

Section: Mouse P-gp Expression In Pichia Pastorismentioning

confidence: 99%

Novel features in the structure of P-glycoprotein (ABCB1) in the post-hydrolytic state as determined at 7.9Å resolution

Thongin

Collins

Barbieri

et al. 2018

Preprint

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P-glycoprotein (ABCB1) is a ATP-binding cassette transporter that plays an important role in the removal of drugs and xenobiotic compounds from the cell.It is also associated with multi-drug resistance in cancer. Here we report novel features of the cryo-EM-derived structure of P-glycoprotein in the posthydrolytic state: The cytosolic nucleotide-binding domains (NBDs) are separated despite ADP remaining bound to the NBDs. Gaps in the TMDs that connect to the inner hydrophilic cavity are back-filled by detergent head-groups from the annular detergent micelle and are close to two regions predicted to delineate two pseudo-symmetry-related drug-binding sites. In this conformation, the (newly-resolved) N-terminal extension, NBD-TMD linker region and gap-filling detergents all appear to impede NBD dimerisation. We propose a model for the mechanism of action of the exporter where ATP will be bound to the protein for most of the time, consistent with the high physiological ATP concentrations in vivo. KeywordsP-glycoprotein; ABCB1; ATP-binding cassette; transporter, membrane protein, protein structure, cryo-electron microscopy.

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Purification of the Cystic Fibrosis Transmembrane Conductance Regulator Protein Expressed in <em>Saccharomyces cerevisiae</em>

Cited by 16 publications

References 26 publications

Specific stabilization of CFTR by phosphatidylserine

Specific stabilization of CFTR by phosphatidylserine

Characterizing diverse orthologues of the cystic fibrosis transmembrane conductance regulator protein for structural studies

Novel features in the structure of P-glycoprotein (ABCB1) in the post-hydrolytic state as determined at 7.9Å resolution

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