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Pet15b vector

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The PET15b vector is a commonly used expression vector for the production of recombinant proteins in Escherichia coli (E. coli) bacterial cells. It contains a T7 promoter for high-level expression of the target gene, as well as an N-terminal His-tag sequence to facilitate purification of the expressed protein.

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Lab products found in correlation

Amicon ultra device, by Merck Group (3 mentions) Escherichia coli bl21 gold de3, by Agilent Technologies (3 mentions) Superdex 75 column, by GE Healthcare (2 mentions) Pgex 4t 1 vector, by GE Healthcare (1 mentions) Ni nitrilotriacetic acid nta column, by Qiagen (1 mentions) Phusion dna polymerase, by Thermo Fisher Scientific (1 mentions) Thrombin, by Cytiva (1 mentions) Pet sumo, by Thermo Fisher Scientific (1 mentions) Blasticidin, by Thermo Fisher Scientific (1 mentions) Pgex 6p 1 vector, by GE Healthcare (1 mentions) Virapower lentiviral expression system, by Thermo Fisher Scientific (1 mentions) His tag purification resin, by Roche (1 mentions) Superdex 75, by GE Healthcare (1 mentions) Pet28b vector, by Merck Group (1 mentions)

11 protocols using pet15b vector

1

Recombinant Protein Purification Techniques

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The gene encoding protein Stl was expressed from a pGEX-4T-1 vector (GE Healthcare, Chicago, Illinois, USA) as a glutathione S-transferase (GST)-fused construct. The GST-tag was cleaved from the protein via overnight thrombin digestion during its purification process [24 (link)]. The E. coli dUTPase gene was inserted into a pET-15b vector (Merck KGaA, Darmstadt, Germany) between the BamHI and NdeI cleavage sites to enable its expression with an N-terminal His-tag which was used in further purification steps [32 (link)]. Both protein Stl and our E. coli dUTPase constructs were expressed in E. coli BL21 (DE3) Rosetta cells (Novagen) and they were purified according to our previously used protocol [24 (link)].
Benedek A., Temesváry-Kis F., Khatanbaatar T., Leveles I., Surányi É.V., Szabó J.E., Wunderlich L, & Vértessy B.G. (2019). The Role of a Key Amino Acid Position in Species-Specific Proteinaceous dUTPase Inhibition. Biomolecules, 9(6), 221.
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2

Purification of Human TFIIH p62 and RPB6

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Unlabeled or 13C/15N-labeled human TFIIH p62 PH-D (residues 1–108) and unlabeled or 13C/15N-labeled human RPB6 (residues 1–127) were prepared as previously described (5 (link)). In brief, p62 or RPB6 was expressed as a hexa-histidine-tagged product in a pET15b vector (Merck Millipore) in Escherichia coli BL21 (DE3) Gold (Agilent Technologies). The lysed supernatant was loaded onto a Ni-nitrilotriacetic acid (NTA) column (QIAGEN), and the eluate was digested with thrombin to remove the histidine tag. After concentration with an Amicon Ultra device (Merck Millipore), the sample was purified on a Superdex75 column (GE Healthcare).
Okuda M., Suwa T., Suzuki H., Yamaguchi Y, & Nishimura Y. (2021). Three human RNA polymerases interact with TFIIH via a common RPB6 subunit. Nucleic Acids Research, 50(1), 1-16.
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3

Engineered Mutations of XylE Transporter

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Full-length wild-type XylE (residues 1-491) gene was PCR amplified from the genomic DNA of E. coli strain BL21(DE3) and subcloned into pET-15b vector (EMD Millipore). Point mutations of full-length XylE were generated using the QuickChange Lightning site-directed mutagenesis protocol (Agilent Technologies). The XylE cytoplasmic domain mutations were generated based on the PCR-driven overlap extension protocol62 using Phusion DNA polymerase (Thermo Scientific). Nucleotides corresponding to residues 232-267 of helices IC2 and IC3 were replaced with nucleotides corresponding to cytoplasmic linker E. coli FucP (residues 237-256) to generate the IC-FucP mutant. The length of the linker was carefully chosen based on superposition analysis of crystal structures of FucP (PDB 3O7Q)5 (link) to match a required distance between end points of the replaced IC2 and IC3 region in XylE in both the outward and inward facing conformations. Nucleotides corresponding to residues 232-244 of helix IC2 were deleted to create the IC2 deletion mutant (delta-IC2). To generate the IC2 sequence scramble mutant (scr-IC2), amino acid residues 232-244 (KQEQAEGILRKIM) were randomly scrambled to the following sequence: ELGIIQKMREKQA. For the IC3 sequence scramble mutant (scr-IC3), amino acid residues 246-266 (NTLATQAVQEIKHSLDHGRKT) were randomly scrambled to the following sequence: TDTHAQLAELTVGSIKHRKQN.
Wisedchaisri G., Park M.S., Iadanza M.G., Zheng H, & Gonen T. (2014). Proton-coupled sugar transport in the prototypical major facilitator superfamily protein XylE. Nature Communications, 5, 4521.
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4

Overexpression and Mutation of BCDIN3D Proteins

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The DNA fragments encoding BCDIN3D proteins from various organisms were synthesized by Eurofins, Japan. The synthesized nucleotide sequences are shown in Supplementary Table S1. For overexpression, the BCDIN3D gene sequences were cloned between the NdeI and XhoI sites of the pET-15b vector (Merck Millipore, Japan). The mutations in the BCDIN3D gene were introduced by the inverse PCR method. The oligonucleotide sequences used for the plasmid constructions are listed in Supplementary Table S2.
Liu Y., Martinez A., Yamashita S, & Tomita K. (2020). Crystal structure of human cytoplasmic tRNAHis-specific 5′-monomethylphosphate capping enzyme. Nucleic Acids Research, 48(3), 1572-1582.
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5

Preparation of Isotope-Labeled spPH Domain

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The 15N- or 13C/15N-labeled spPH domain (residues 1-108) was prepared by a previously described method [28 (link)]. In brief, the spPH domain was expressed as a hexa-histidine-tagged product in a pET15b vector (Merck Millipore) in Escherichia coli BL21-Gold (DE3) (Agilent Technologies). The lysed supernatant was loaded on to a cOmplete His-Tag purification resin column (Roche), and the eluate was digested with thrombin (Cytiva) to remove the histidine tag. After concentration with an Amicon Ultra device (Merck Millipore), the sample was purified on a Superdex75 column (GE Healthcare).
Okuda M, & Nishimura Y. (2023). Structural polymorphism of the PH domain in TFIIH. Bioscience Reports, 43(7), BSR20230846.
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6

Construction of Fluorescent Protein Plasmids

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pET15-sfGFP, pET29-sfGFP were prepared in previous studies (Fujiwara and Nomura, 2013 (link); Fujiwara and Doi, 2016 (link)). pET15-TagBFP2, pET29-mCherry-His, pET29-αHL were constructed in this study. pET15-TagBFP2 was constructed by ligation of NdeI/XhoI-digested fragments of pET15b vector (Merck Millipore, Billerica, MA, USA) and a synthesized gene of TagBFP2 (Thermo Fisher Scientific). pET29-mCherry-His was constructed by removing minE gene from pET29-MinE-mCherry-His (Kohyama et al., 2019 (link)) by PCR and a primer set (pET29mCherryFw/pET29u). pET29-αHL was constructed by fusing pET29 vector amplified by a primer set (pET29d/pET29u) with C-terminal histidine-tagged αHL gene (Fujii et al., 2013 (link)) prepared by PCR. PCR fragments were converted to plasmids by SLIC method. To attach DNA histidine-tag to αHL gene, two times PCR was performed using primer sets (aHLfw/aHLrv1 for 1st PCR, aHLfw/aHLrv1 for 2nd PCR). PrimeSTAR MAX DNA polymerase was used for all PCR, and all DNA sequences were verified by Sanger DNA sequencing. Primer sequences are shown in Table S2.
Akui T., Fujiwara K., Sato G., Takinoue M., Nomura S.I, & Doi N. (2021). System concentration shift as a regulator of transcription-translation system within liposomes. iScience, 24(8), 102859.
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7

Plasmid Acquisition and Characterization

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The Zero Blunt PCR vector was obtained from Thermo Fisher Scientific, the pET15b vector was obtained from EMD Millipore (Billerica, MA, USA), the pHis-Trx vector was kindly provided by Professor Andrea Brancaccio from the Consiglio Nazionale delle Richerche, and pET-SUMO was obtained from Thermo Fisher Scientific. Other chemical reagents were of analytical grade.
Bugli F., Caprettini V., Cacaci M., Martini C., Paroni Sterbini F., Torelli R., Della Longa S., Papi M., Palmieri V., Giardina B., Posteraro B., Sanguinetti M, & Arcovito A. (2014). Synthesis and characterization of different immunogenic viral nanoconstructs from rotavirus VP6 inner capsid protein. International Journal of Nanomedicine, 9, 2727-2739.
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8

Generation and Characterization of Grp1 Constructs

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Myc-tagged mouse Grp1 (wild-type and T280D) in pENTR and pCDNA3.1 that are siRNA resistant have been described (Li et al., 2012 (link)). A PI4P biosensor, known as GFP-P4M-SidM, has also been described (Hammond et al., 2014 ), and was obtained from Addgene (plasmid #51469). 6xHis-tagged GRASP and IPCEF1 were generated by subcloning the coding sequence of GRASP and IPCEF1 into pET-15b vector (EMD Millipore, Billerica, MA). To generate GST fusions of Grp1, GRASP, and IPCEF1, the coding sequences of proteins were amplified by PCR, and then subcloned into pGEX-6P-1 vector (GE Healthcare, Pittsburgh, PA). Different forms of Myc-tagged Grp1 were subcloned and inserted into pENTR (Invitrogen, Carlsbad, CA). Point mutants were then generated by a QuikChange II XL site-directed mutagenesis kit (Stratagene, La Jolla, CA). Myc-Grp1 constructs in pLenti6.2 were further generated by recombination according to the Gateway protocol provided (Invitrogen). Lentiviral particles expressing Myc-Grp1 constructs were generated using a ViraPower Lentiviral Expression System (Invitrogen). 3T3-L1 fibroblast cell lines that stably express different forms of Myc-Grp1 were generated by lentiviral transduction followed by selection in 10 μg/ml blasticidin (Invitrogen).
Li J., Lambright D.G, & Hsu V.W. (2020). Coordination of Grp1 recruitment mechanisms by its phosphorylation. Molecular Biology of the Cell, 31(25), 2816-2825.
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9

Expression and Purification of 13C/15N-labeled p62 Fragment

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The 13C/15N-labeled fragment of human p621–158 (residues 1–158) was expressed as a hexa-histidine-tagged product in a pET15b vector (Merck Millipore) in Escherichia coli BL21 (DE3) Gold (Agilent Technologies). The cells were grown at 37°C in M9 minimal medium containing [15N]ammonium chloride and [13C]glucose. The product was expressed by induction with 1 mM isopropyl-β-d-thiogalactopyranoside. After 21 h of growth at 20°C, the cells were collected, resuspended in buffer A [20 mM Tris–HCl (pH 8.0), 10% glycerol, 1 M NaCl], lysed by sonication and then centrifuged. The supernatant was loaded onto a His-Tag Purification Resin (Roche) column equilibrated with buffer A, and the column was washed with buffer A containing 10 mM imidazole–HCl. The sample was eluted by 500 mM imidazole–HCl, peak fractions were pooled and the buffer was changed to thrombin cleavage buffer (10 mM Na2HPO4, 1.8 mM KH2PO4, 500 mM NaCl, 2.7 mM KCl, pH 7.3) by using an Amicon Ultra device (Merck Millipore). To remove the histidine tag, the sample was digested with thrombin for 63 h at 20°C. After concentration via an Amicon Ultra, the sample was applied to a Superdex75 (GE Healthcare) column equilibrated with 20 mM potassium phosphate (pH 6.8) and 500 mM NaCl.
Okuda M., Ekimoto T., Kurita J.I., Ikeguchi M, & Nishimura Y. (2020). Structural and dynamical insights into the PH domain of p62 in human TFIIH. Nucleic Acids Research, 49(5), 2916-2930.
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10

Cloning and Expression of Ion Channels

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The KcsA channel gene was cloned into the pET28b(+) vector [EMD Millipore] for CFPS and E. coli expression.18 (link) Additionally, CFPS of the KcsA channel was also carried out with the gene cloned into the pET15b vector [EMD Millipore]. A synthetic gene for the MVP channel that was codon optimized for E. coli expression was kindly provided by Dr. Steve Goldstein (Brandeis University).19 (link) The MVP channel gene was cloned into the pET28b(+) vector for CFPS and for E. coli expression. The LeuT transporter gene cloned in the pET16b vector was kindly provided by Dr. Eric Gouaux (Vollum Institute, OHSU).20 (link) The LeuT gene was transferred to the pET28b(+) vector for CFPS.
Focke P.J., Hein C., Hoffmann B., Matulef K., Bernhard F., Dötsch V, & Valiyaveetil F.I. (2016). Combining In vitro Folding with Cell-Free Protein Synthesis for Membrane Protein Expression. Biochemistry, 55(30), 4212-4219.
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