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Pdisplay bira er

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The PDisplay-BirA-ER is a plasmid that can be used to biotinylate proteins within the endoplasmic reticulum of cells. The plasmid encodes the BirA enzyme, which catalyzes the attachment of biotin to proteins containing a specific peptide sequence.

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Lab products found in correlation

Expi293f cells, by Thermo Fisher Scientific (2 mentions) Superdex 75 10 300 gl gel filtration column, by GE Healthcare (2 mentions) Histrap nickel column, by GE Healthcare (2 mentions) Freestyle 293 expression medium, by Thermo Fisher Scientific (2 mentions) D biotin, by Merck Group (2 mentions) Lipofectamine 2000, by Thermo Fisher Scientific (2 mentions) Emerin pegfp n2 588, by Addgene (1 mentions) Pcdna3 connexin43 gfp apex2, by Addgene (1 mentions) Pegfp n1, by Addgene (1 mentions) Protease and phosphatase inhibitor, by Cell Signaling Technology (1 mentions) 6 well culture dishes, by Greiner (1 mentions) Poly prep column, by Bio-Rad (1 mentions) Fugene hd transfection reagent, by Promega (1 mentions) Gblock gene fragment, by Integrated DNA Technologies (1 mentions) Biacore t200, by GE Healthcare (1 mentions)

6 protocols using pdisplay bira er

1

Engineered Connexin43 Constructs for Cellular Analysis

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The ER-retention motif RRRRISLS was subcloned into a pENTR-V5-Cx43 vector to generate Cx43RRRRISLS. Plasmids expressing GFPCx43, GFPmCherryCx43, Cx43Avi and BirA-NLS were generated by cloning the appropriate cDNA into a pENTR vector containing the indicated tags. Plasmids expressing Cx43ΔNLS were generated by amplifying cDNA encoding the first 236 and last 91 amino acids of rat Cx43, followed by a second PCR to unite the fragments; and finally cloned into a pENTR V5 vector. NLS-EGFP was kindly provided by Rob Parton (Addgene #67652) [69 (link)], mEmerald-Nesprin3-C-18 from Michael Davidson (Addgene #54203), Emerin pEGFP-N2 (588) from Eric Schirmer (Addgene #61985) [70 (link)], pDESTmycYBX1 from Thomas Tuschl (Addgene #19878) [71 (link)], pEGFP-C2 RanGAP from Mary Dasso (Addgene #13378) [72 (link)], pEGFP-N1 full length Importin- β was a gift from Patrizia Lavia (Addgene #106941) [73 (link)], pcDNA3 Connexin43-GFP-APEX2 (Addgene #49385) and pDisplay-BirA-ER (Addgene #20856) from Alice Ting [74 (link),75 (link)]. Experiments were performed 24 h after transient transfection of cells with Lipofectamine 2000 (Thermo Fisher Scientific), according to the manufacturer.
Martins-Marques T., Witschas K., Ribeiro I., Zuzarte M., Catarino S., Ribeiro-Rodrigues T., Caramelo F., Aasen T., Carreira I.M., Goncalves L., Leybaert L, & Girao H. (2023). Cx43 can form functional channels at the nuclear envelope and modulate gene expression in cardiac cells. Open Biology, 13(11), 230258.
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2

Transfection and Purification of MET Constructs

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pIRESneo-MET or pIRESneo-METΔ7–8 were transfected into HEK-293T or TOV-112D cells in 6-well culture dishes (Greiner Bio-One, Krëmsmunster, Austria) using Fugene HD transfection reagent (Promega, Fitchburg, WI, USA) according to the manufacturers’ instructions. After 48 h, cell monolayers were washed with PBS and cell extracts prepared in RIPA buffer containing protease and phosphatase inhibitors (Cell Signaling Technology, CST, Danvers, MA, USA).
In separate experiments, pHLsec-METΔ7–8ED-BAPHIS or pHLsec-METED-BAPHIS were cotransfected in a 1:1 ratio with the biotin-ligase expression construct pDISPLAY-BirA-ER (Addgene) in HEK293T cells and cells were cultured in the presence of 10 μM biotin (Sigma-Aldrich, St Louis, MO, USA). After 48 h, cytosolic extracts were made in RIPA buffer, and medium (1 ml) was mixed with 100 μl Ni–NTA Sepharose slurry (IBA, Goettingen, Germany). After a 1 h incubation at 4 °C, Ni-beads were washed with buffer (500 mM NaCl, 50 mM phosphate buffer, pH 7.4) and loaded onto a poly-prep column (Bio-Rad, Hercules, CA, USA). After washing off specifically bound proteins with 2 ml 10 mM imidazole, His-tagged an biotinylated MET extracellular domains were eluted with 0.5 ml 0.5 M imidazole, and dialysed o/n at 4 °C to 50 mM TRIS/150 mM NaCl pH 7.5.
Navis A.C., van Lith S.A., van Duijnhoven S.M., de Pooter M., Yetkin-Arik B., Wesseling P., Hendriks W.J., Venselaar H., Timmer M., van Cleef P., van Bergen en Henegouwen P., Best M.G., Wurdinger T.D., Tops B.B, & Leenders W.P. (2015). Identification of a novel MET mutation in high-grade glioma resulting in an auto-active intracellular protein. Acta Neuropathologica, 130(1), 131-144.
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3

AviTag-GluR2/eGFP/BirA Plasmid Construction

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The AviTag-GluR2/eGFP/BirA plasmid used in this study was created through modification of an AviTag-GluR2 plasmid. A gBlock Gene Fragment (Integrated DNA Technologies) encoding eGFP and a T2A peptide under control of an EF1a core promoter (EFS) was ligated into the backbone through Gibson assembly. A DNA fragment encoding an IgK leader followed by BirA was generated by PCR from pDisplay-BirA-ER (Addgene plasmid #20856) and cloned in frame with eGFP and the T2A peptide through Gibson assembly. The plasmid sequence is included in the Supporting Methods.
Le P., Vaidya R., Smith L.D., Han Z., Zahid M.U., Winter J., Sarkar S., Chung H.J., Perez-Pinera P., Selvin P.R, & Smith A.M. (2020). Optimizing Quantum Dot Probe Size for Single-Receptor Imaging. ACS nano, 14(7), 8343-8358.
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4

Recombinant SARS-CoV-2 RBD Production

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Plasmids encoding RBDs were transfected into Expi293F Cells (ThermoFisher) by PEI, cultured in FreeStyle 293 Expression Medium (ThermoFisher) at 37°C for 1 day followed by 30°C for 3 days with 8% CO2. To express biotinylated RBDs, the RBD-BAP plasmid was co-transfected with pDisplay-BirA-ER (Addgene plasmid 20,856; coding for an ER-localized biotin ligase), in the presence of 0.8 mM D-biotin (Sigma-Aldrich). The conditioned medium was diluted 1:2 into binding buffer (50 mM sodium phosphate, 500 mM sodium chloride, pH 8.0). RBDs were purified with a 5 mL HisTrap nickel column (GE Healthcare) through His-tag binding, followed by a Superdex 75 10/300 GL gel filtration column (GE Healthcare) in 10 mM HEPES and 150 mM sodium chloride.
Huo J., Dijokaite-Guraliuc A., Liu C., Zhou D., Ginn H.M., Das R., Supasa P., Selvaraj M., Nutalai R., Tuekprakhon A., Duyvesteyn H.M., Mentzer A.J., Skelly D., Ritter T.G., Amini A., Bibi S., Adele S., Johnson S.A., Paterson N.G., Williams M.A., Hall D.R., Plowright M., Newman T.A., Hornsby H., de Silva T.I., Temperton N., Klenerman P., Barnes E., Dunachie S.J., Pollard A.J., Lambe T., Goulder P., Fry E.E., Mongkolsapaya J., Ren J., Stuart D.I, & Screaton G.R. (2022). A delicate balance between antibody evasion and ACE2 affinity for Omicron BA.2.75. Cell Reports, 42(1), 111903.
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5

Immobilization and Biotinylation of Proteins

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cDNA for the immobilized proteins (CPTX, NP1PTX, Cbln1) was cloned into the pHLsec-Avitag3 vector (61 (link)), resulting in proteins carrying a C-terminal biotin ligase (BirA) recognition sequence (Avitag). Constructs were co-transfected with pDisplay-BirA-ER (Addgene cat. # 20856; coding for an ER-resident biotin ligase) (78 (link)) for in vivo biotinylation in HEK293T cells in small-scale 6-well plates in a 3:1 pHLsec:pDisplay stoichiometric ratio. A concentration of 100 µM D-biotin was maintained in the expression medium to ensure biotinylation of the Avitag. After 48 h of expression, conditioned medium was collected and dialyzed against 10 mM Tris pH 7.4, 150 mM sodium chloride, 3 mM calcium chloride and 0.005% (v/v) Tween-20 (TBS-CT). SPR experiments were performed on a Biacore T200 (GE Healthcare) operated at a data collection frequency of 10 Hz. Streptavidin (Sigma-Aldrich cat. # S4762) was chemically coupled via amine coupling chemistry onto CM5 chips to a response unit (RU) level of 5000 RU. Then, biotinylated proteins were captured to the desired RU level. In each instance, for every two analyte binding cycles, a buffer injection was performed, allowing for double referencing of the binding responses (79 (link)).
Suzuki K., Elegheert J., Song I., Sasakura H., Senkov O., Matsuda K., Kakegawa W., Clayton A.J., Chang V.T., Ferrer-Ferrer M., Miura E., Kaushik R., Ikeno M., Morioka Y., Takeuchi Y., Shimada T., Otsuka S., Stoyanov S., Watanabe M., Takeuchi K., Dityatev A., Aricescu A.R, & Yuzaki M. (2020). A synthetic synaptic organizer protein restores glutamatergic neuronal circuits. Science (New York, N.Y.), 369(6507), eabb4853.
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6

Recombinant SARS-CoV-2 RBD Expression

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Plasmids encoding RBDs were transfected into Expi293F™ Cells (ThermoFisher) by PEI, cultured in FreeStyle™ 293 Expression Medium (ThermoFisher) at 30 °C with 8% CO2 for 4 days. To express biotinylated RBDs, the RBD-BAP plasmid was co-transfected with pDisplay-BirA-ER (Addgene plasmid 20856; coding for an ER-localized biotin ligase), in the presence of 0.8 mM D-biotin (Sigma-Aldrich). The conditioned medium was diluted 1:2 into binding buffer (50 mM sodium phosphate, 500 mM sodium chloride, pH 8.0). RBDs were purified with a 5 mL HisTrap nickel column (GE Healthcare) through His-tag binding, followed by a Superdex 75 10/300 GL gel filtration column (GE Healthcare) in 10 mM HEPES and 150 mM sodium chloride.
Tuekprakhon A., Nutalai R., Dijokaite-Guraliuc A., Zhou D., Ginn H.M., Selvaraj M., Liu C., Mentzer A.J., Supasa P., Duyvesteyn H.M., Das R., Skelly D., Ritter T.G., Amini A., Bibi S., Adele S., Johnson S.A., Constantinides B., Webster H., Temperton N., Klenerman P., Barnes E., Dunachie S.J., Crook D., Pollard A.J., Lambe T., Goulder P., Paterson N.G., Williams M.A., Hall D.R., Fry E.E., Huo J., Mongkolsapaya J., Ren J., Stuart D.I, & Screaton G.R. (2022). Antibody escape of SARS-CoV-2 Omicron BA.4 and BA.5 from vaccine and BA.1 serum. Cell, 185(14), 2422-2433.e13.
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