Recombinant Human CFTR

Cat.No. : CFTR-27241TH
Product Overview : Recombinant fragment of Human CFTR (amino acids 1381-1480) with N terminal proprietary tag, 36.63kDa.
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Species : Human
Source : Wheat Germ
Tag : Non
Protein Length : 100 amino acids
Description : This gene encodes a member of the ATP-binding cassette (ABC) transporter superfamily. ABC proteins transport various molecules across extra- and intra-cellular membranes. ABC genes are divided into seven distinct subfamilies (ABC1, MDR/TAP, MRP, ALD, OABP, GCN20, White). This protein is a member of the MRP subfamily that is involved in multi-drug resistance. The encoded protein functions as a chloride channel and controls the regulation of other transport pathways. Mutations in this gene are associated with the autosomal recessive disorders cystic fibrosis and congenital bilateral aplasia of the vas deferens. Alternatively spliced transcript variants have been described, many of which result from mutations in this gene.
Molecular Weight : 36.630kDa inclusive of tags
Tissue specificity : Found on the surface of the epithelial cells that line the lungs and other organs.
Form : Liquid
Purity : Proprietary Purification
Storage buffer : pH: 8.00Constituents:0.3% Glutathione, 0.79% Tris HCl
Storage : Shipped on dry ice. Upon delivery aliquot and store at -80oC. Avoid freeze / thaw cycles.
Sequences of amino acids : YQIIRRTLKQAFADCTVILCEHRIEAMLECQQFLVIEENKVRQYDSIQKLLNERSLFRQAISPSDRVKLFPHRNSSKCKSKPQIAALKEETEEEVQDTRL
Sequence Similarities : Belongs to the ABC transporter superfamily. ABCC family. CFTR transporter (TC 3.A.1.202) subfamily.Contains 2 ABC transmembrane type-1 domains.Contains 2 ABC transporter domains.
Gene Name CFTR cystic fibrosis transmembrane conductance regulator (ATP-binding cassette sub-family C, member 7) [ Homo sapiens ]
Official Symbol CFTR
Synonyms CFTR; cystic fibrosis transmembrane conductance regulator (ATP-binding cassette sub-family C, member 7); ABCC7, CF, cystic fibrosis transmembrane conductance regulator, ATP binding cassette (sub family C, member 7); cystic fibrosis transmembrane conducta
Gene ID 1080
mRNA Refseq NM_000492
Protein Refseq NP_000483
MIM 602421
Uniprot ID P13569
Chromosome Location 7q31-q32
Pathway ABC transporters, organism-specific biosystem; ABC transporters, conserved biosystem; ABC-family proteins mediated transport, organism-specific biosystem; Bile secretion, organism-specific biosystem; Bile secretion, conserved biosystem;
Function ATP binding; ATP-binding and phosphorylation-dependent chloride channel activity; ATPase activity; PDZ domain binding; channel-conductance-controlling ATPase activity;

Cystic fibrosis transmembrane conductance regulator attaches tumor suppressor PTEN to the membrane and promotes anti Pseudomonas aeruginosa immunity

Journal: Immunity    PubMed ID: 29246444    Data: 2018/12/19

Authors: Sebastián A. Riquelme, Benjamin D. Hopkins, Alice Prince

Article Snippet:Chemicals, Peptides, and Recombinant Proteins , , .Chemicals, Peptides, and Recombinant Proteins , , .. Recombinant Human CFTR (1381-1480), GST-tagged , Creative-BioMart , Cat# CFTR-275H Lot # 101018.. Recombinant Human CFTR (1381-1480), GST-tagged , Abnova , Cat# H00001080-Q01 Lot # G7151.Recombinant Human CFTR (1381-1480), GST-tagged , Abnova , Cat# H00001080-Q01 Lot # G7151.

(A) Frontal and Lateral (left) view of the crystallized non-phosphorylated human CFTR and PTEN. PTEN PDB that represents crystalized PTEN does not show the PTEN C-terminus (PTEN14-351), which has been demonstrated as a negative regulator for its interaction with the membrane. Different CFTR domains are shown in different colors (MSD1-2: membrane-spanning domain; NBD1-2: nucleotide-binding domain 1–2; R: regulatory domain). CFTRC-term tail (CFTR1381-1440) is shown in red at the end of NBD2 (red arrow). PTEN (gray) and CFTR-NBD2 PDB structures where submitted to ClusProV2 clustering and modelling. (B) Model 1 (out of 4, see Figure S5A) from ClusProV2 protein-protein interaction modelling showing the interaction between PTEN14-351 and CFTR-NBD2 (CFTR1207-1440). The whole human CFTR structure complexed with human PTEN is showed to better display the orientation of PTEN from the cytoplasm. (C) Human crystallized CFTR-NBD2 and PTEN interaction model 1 from ClusProV2. CFTR1207-1380 is shown in purple. CFTR1381-1440 is shown in red. PTEN is shown in gray. Amino acids from CFTR1207-1440 taking contact with PTEN are displayed in cyan. PTEN amino acids contacting CFTR1207-1440 are shown in orange. (D) I-TASSER modelled CFTR1381-1480 and PTEN interaction model from ClusProV2. CFTR1381-1440 is shown in red. CFTR1441-1480 is shown in green. PTEN is shown in gray. Amino acids from CFTR1381-1480 taking contact with PTEN are displayed in cyan. PTEN amino acids contacting CFTR1381-1480 are shown in orange. (E) ELISA plates were coated either with rGST alone or rGST-CFTR1381-1480. Wells were blocked, washed and incubated with PTEN-containing human 16HBE cell lysates. Plates were revealed for PTEN. (F) ELISA plates were coated either with Vehicle or rPTEN lacking the C-terminus (PTEN1-351). Wells were blocked, washed and incubated with CFTR-containing human 16HBE cell lysates. Plates were revealed for CFTR. (G) CFTR-PTEN and CFTRCterm-PTEN proximity ligation assays (PLA) in human epithelial cells (16HBE). Intracellular detection of CFTR was performed by using either an antibody against a naturally extracellular-exposed loop of CFTR (anti-CFTR) or the CFTRC-term (anti-CFTRC-term). Control cells did not receive primary antibodies. Zoom insets show protein-protein interaction clusters. (H) Quantification of the amount of CFTR-PTEN or CFTRC-term-PTEN clusters obtained with PLA. (I) Pull down analyses between either recombinant CFTRC-term (rGST-CFTR1381-1480) or rGST with rPTEN lacking the C-terminus (PTEN1-351). PTEN was N-terminus tagged with a His-Tag. Anti-GST and anti-His were used to detect both GST-CFTR1381-1480, GST and His-rPTEN, respectively. (J) Human PBMCs from both WT (CFTR1- 1480) and W1282X mutants CF patients (CFTR1-1281) which lack CFTRC-term were stained for PTEN and analyzed by flow cytometry. (K) Human monocytes were exposed to GFP-expressing P. aeruginosa (PAK, shown as cyan in this picture) (MOI=10) and stained for CFTR (green), PTEN (red) and nuclei (blue). Where indicated, R1, R2 and R3 are region of interest (ROI) zoomed at the right part of the main figure. Colocalization between CFTR and PTEN can be seen as yellow color in insets. Only one confocal plane is shown. Data was analyzed by using FIJI. White bar represents 3μmts. Image represents 2 independent experiments. K represent 4 and E, F and H represent 3 independent experiments, respectively. Data is presented as average ± SEM. E–F were analyzed by Two-Ways ANOVA. H was analyzed by one-way ANOVA. For J, data were analyzed by t-Student test. *, p<0.05; ***, p<0.001; ****, p<0.0001; ns: non-significant. See also Figure S5.

(A) Frontal and Lateral (left) view of the crystallized non-phosphorylated human CFTR and PTEN. PTEN PDB that represents crystalized PTEN does not show the PTEN C-terminus (PTEN14-351), which has been demonstrated as a negative regulator for its interaction with the membrane. Different CFTR domains are shown in different colors (MSD1-2: membrane-spanning domain; NBD1-2: nucleotide-binding domain 1–2; R: regulatory domain). CFTRC-term tail (CFTR1381-1440) is shown in red at the end of NBD2 (red arrow). PTEN (gray) and CFTR-NBD2 PDB structures where submitted to ClusProV2 clustering and modelling. (B) Model 1 (out of 4, see Figure S5A) from ClusProV2 protein-protein interaction modelling showing the interaction between PTEN14-351 and CFTR-NBD2 (CFTR1207-1440). The whole human CFTR structure complexed with human PTEN is showed to better display the orientation of PTEN from the cytoplasm. (C) Human crystallized CFTR-NBD2 and PTEN interaction model 1 from ClusProV2. CFTR1207-1380 is shown in purple. CFTR1381-1440 is shown in red. PTEN is shown in gray. Amino acids from CFTR1207-1440 taking contact with PTEN are displayed in cyan. PTEN amino acids contacting CFTR1207-1440 are shown in orange. (D) I-TASSER modelled CFTR1381-1480 and PTEN interaction model from ClusProV2. CFTR1381-1440 is shown in red. CFTR1441-1480 is shown in green. PTEN is shown in gray. Amino acids from CFTR1381-1480 taking contact with PTEN are displayed in cyan. PTEN amino acids contacting CFTR1381-1480 are shown in orange. (E) ELISA plates were coated either with rGST alone or rGST-CFTR1381-1480. Wells were blocked, washed and incubated with PTEN-containing human 16HBE cell lysates. Plates were revealed for PTEN. (F) ELISA plates were coated either with Vehicle or rPTEN lacking the C-terminus (PTEN1-351). Wells were blocked, washed and incubated with CFTR-containing human 16HBE cell lysates. Plates were revealed for CFTR. (G) CFTR-PTEN and CFTRCterm-PTEN proximity ligation assays (PLA) in human epithelial cells (16HBE). Intracellular detection of CFTR was performed by using either an antibody against a naturally extracellular-exposed loop of CFTR (anti-CFTR) or the CFTRC-term (anti-CFTRC-term). Control cells did not receive primary antibodies. Zoom insets show protein-protein interaction clusters. (H) Quantification of the amount of CFTR-PTEN or CFTRC-term-PTEN clusters obtained with PLA. (I) Pull down analyses between either recombinant CFTRC-term (rGST-CFTR1381-1480) or rGST with rPTEN lacking the C-terminus (PTEN1-351). PTEN was N-terminus tagged with a His-Tag. Anti-GST and anti-His were used to detect both GST-CFTR1381-1480, GST and His-rPTEN, respectively. (J) Human PBMCs from both WT (CFTR1- 1480) and W1282X mutants CF patients (CFTR1-1281) which lack CFTRC-term were stained for PTEN and analyzed by flow cytometry. (K) Human monocytes were exposed to GFP-expressing P. aeruginosa (PAK, shown as cyan in this picture) (MOI=10) and stained for CFTR (green), PTEN (red) and nuclei (blue). Where indicated, R1, R2 and R3 are region of interest (ROI) zoomed at the right part of the main figure. Colocalization between CFTR and PTEN can be seen as yellow color in insets. Only one confocal plane is shown. Data was analyzed by using FIJI. White bar represents 3μmts. Image represents 2 independent experiments. K represent 4 and E, F and H represent 3 independent experiments, respectively. Data is presented as average ± SEM. E–F were analyzed by Two-Ways ANOVA. H was analyzed by one-way ANOVA. For J, data were analyzed by t-Student test. *, p<0.05; ***, p<0.001; ****, p<0.0001; ns: non-significant. See also Figure S5.

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