pET30a-NS1-70His and pET30a-Ku70Strep were constructed by inserting NS1-70 open reading frame (ORF) or Ku70 ORF into the pET30a(+) vector (number 69909; MilliporeSigma) between NdeI and XhoI sites. The Ku70 domains expressing pET30a vectors were constructed by inserting Ku70 amino acid 1 to 250, 251 to 440, 437 to 609, 437 to 534, and 535 to 609 coding sequences between the NdeI and XhoI sites for domains A, B, C, C-Arm, and SAP. All constructs expressed the Strep II tag at the C terminus of the target proteins.
Pet30a vector
Manufactured by Merck Group
Sourced in Germany
The PET30a vector is a plasmid used for protein expression in Escherichia coli. It contains the T7 promoter, a multiple cloning site, and a kanamycin resistance gene for selection. The vector allows for the production of recombinant proteins in bacterial systems.
Automatically generated - may contain errors
Lab products found in correlation
Abi 3730, by Thermo Fisher Scientific (3 mentions)
Big dye chemistry, by Thermo Fisher Scientific (2 mentions)
Rosetta 2 de3 plyss competent cells, by Merck Group (2 mentions)
Bigdye, by Thermo Fisher Scientific (1 mentions)
Quikchange mutagenesis kit, by Agilent Technologies (1 mentions)
Nucleospin plasmid dna kit, by Macherey-Nagel (1 mentions)
Phusion dna polymerase, by New England Biolabs (1 mentions)
Aminolink plus coupling resin, by Thermo Fisher Scientific (1 mentions)
Ni nta his bind resin, by Merck Group (1 mentions)
Histrap hp column, by GE Healthcare (1 mentions)
Ez link sulfo nhs ss biotin, by Thermo Fisher Scientific (1 mentions)
Histalon column, by Takara Bio (1 mentions)
Ni nta agarose, by Qiagen (1 mentions)
Lightshift chemiluminescent emsa kit, by Thermo Fisher Scientific (1 mentions)
18 protocols using pet30a vector
1
Constructing pET30a Protein Expression Vectors
2
Cloning and Expressing Aat1 Protein
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Full length Aat1 transcript (NCBI Reference Sequence NP_001148286.2) was amplified using PCR with primers 5′-CATGATTCGAATTC ATGGCGGCAGCAACGGCAACT-3′ and 5′-GCTACGATAAGCTT TCACAGCAACCGGAGCCACTCCA-3′ that introduce EcoRI and HindIII sites (underlined). Restriction enzyme cloning introduced the transcripts into the pET-30a(+) vector (Millipore Sigma, Burlington, MA, USA) as described by the supplier [24 ]. Vectors were transformed into chemically competent Rosetta-gami™ 2(DE3)pLysS Competent Cells (Millipore Sigma, Burlington, MA, USA). LB media supplemented with 1 mM MgSO4 to assist growth [25 (link)] and 50 µg/mL kanamycin for selection were inoculated with cultures stored at −80 °C in 25% (v/v) glycerol. After cultivating overnight at 37 °C, two mL of overnight culture was added to 100 mL fresh media and grown at 37 °C to an OD600 of 0.6 to 0.8. Cultures were put on ice for five minutes before being induced with 0.4 mM IPTG overnight at 18 °C. Overnight cultures were centrifuged at 1600× g for 10 min. Media were decanted and pellets were resuspended in 100 mM potassium phosphate (pH 8.0), 10% glycerol, 1 mM EDTA, and 5 mM 2-mercaptoethanol (Buffer A) with an addition of 1 mM PMSF. Bacterial lysis by sonication was done on ice with a 20 kHz probe set to 50% power with five 10 s bursts with 20 s rests between pulses.
3
Recombinant VEGFA-Fc Fusion Protein
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The polybasic tag (PBT) sequence was derived from human VEGFA, protein sequence accession: NP_001020537.2, amino acids 131 - 163. The Fc fragment was derived from human IgG1/IGHG1 heavy chain sequence from amino acids 239 - 470, and was reported previously 21 (link). The PBT and Fc modules were joined by in-frame fusion of their encoding cDNA fragments, which were cloned into pET-30a vector (Millipore-Sigma) with an N-terminus 6xHis tag as an extension. The Fc alone fragment was also cloned into pET-30a vector as control.
4
Tau Protein Mutant Generation
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MARK2/Par1-myc was kindly provided by B. Lu (Stanford University School of Medicine, Stanford, CA), whereas GFP and wild-type V5-tagged tau (4R0N isoform) constructs for mammalian expression were cloned into pcDNA3.1 in our laboratory (31 (link), 36 (link)). The QuikChange mutagenesis kit (Agilent Technologies, Santa Clara, CA) was used to generate K163Q, K174Q, K180Q, K234Q, K240Q, K259Q, K274Q, K280Q, K290Q, K311Q, K321Q, K353Q, K369Q, K290Q/K321Q, K259Q/K290Q/K321Q/K353Q, K321R, S324A, S324D, and S324E mutants from the wild-type tau-V5 parent construct, following the manufacturer's protocol. For protein expression in bacteria, wild-type and mutant tau constructs were cloned into the pET30a vector (EMD Millipore, Billerica, MA). All constructs were sequence-verified using ABI3730 with Big Dye chemistry following the manufacturer's protocols (Applied Biosystems, Foster City, CA).
5
Bacterial Culturing for Genetic Manipulation
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All bacterial cultures were grown under aerobic conditions at 37°C with shaking at 220 rpm in Luria-Bertani (LB) medium supplemented with chloramphenicol (50 µg/ml), kanamycin (30 µg/ml), ampicillin (100 µg/ml), and streptomycin (50 µg/ml) as required for plasmid maintenance. The pET30a (+) vector was purchased from EMD Millipore (Billerica, MA). R. sphaeroides 2.4.1 and C. tepidum TLS were acquired from the ATCC collection (Manassas, VA). E. coli JM109 was used for all genetic manipulations and E. coli BL21 (DE3) was used for protein expression.
6
Recombinant Tau and Alpha-Synuclein Proteins
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Human cDNAs encoding full-length wild-type tau (4R0N isoform), AD tau core, CBD tau core, PiD tau core, K18 tau fragment, and alpha-synuclein were all cloned into the pET30a vector (EMD Millipore) and transformed into Rosetta 2(DE3) pLysS competent cells (EMD Millipore) for protein expression. All constructs were sequence-verified using ABI3730 with Big Dye chemistry following manufacturer’s protocols (Applied Biosystems, Foster City, CA, USA).
7
Recombinant Tau and Alpha-Synuclein Proteins
8
RBD Mutant Protein Production
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The pET30a vector (Novagen, Merck Group) encoding for the RBD fused with an N-terminal superfolder GFP (sfGFP) fusion tag (Pedelacq et al. 2006 (link)) and a C-terminal His tag was used as the template for site-directed mutagenesis. The amino acid residues 442–460 and 473–495 in the RBD were individually replaced with alanine by using the PCR-based site-directed mutagenesis method as described in the previous study (Lai et al. 2022 (link)). All mutations were confirmed by Sanger sequencing.
9
RBD Mutant Protein Expression and Characterization
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The cDNA encoding for RBD was subcloned into the pET30a vector (Novagen, Merck Group) with a C-terminal His-tag and an N-terminal superfolder GFP (sfGFP) fusion tag to increase the protein solubility (Pedelacq et al., 2006 (link)). The pET30a-sfGFP-RBD-His plasmid was used as the template for site-directed mutagenesis. The codons encoding for the amino acid residues 477–495 in the RBD were substituted individually with the codons of alanine by using the PCR-based site-directed mutagenesis method. All mutations were confirmed by Sanger sequencing. The vectors for expressing the sfGFP-RBD-His mutants were transformed into E. coli BL21(DE3) competent cells, and the protein expression were induced as described previously. Bacterial cells were collected for protein extraction, which was then subjected to SDS-PAGE and WB analysis with the anti-His tag antibody and 1Ba–3H.
10
Cloning of the agltp24 Gene
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The agltp24 gene was PCR amplified from cDNA prepared above using designed primers (Table S1) under the following conditions: 98 °C for 30 s; 30 cycles of 98 °C for 7 s, 63 °C for 20 s and 72 °C for 8 s. The reaction mixture of 50 µl PCR contained 1X Phusion HF buffer, 200 µM dNTPs, 0.5 µM Forward primer, 0.5 µM reverse primer, 87 ng template DNA and 1-units Phusion DNA Polymerase (NEB, Evry, France). The PCR product was cloned using the hot fusion method (Fu et al., 2014) in pET30a+ vector (Merck, Molsheim France) pre-digested with EcoRI and BglII in order to fusion AgLTP24 with a N-terminal 6XHis flag. Ligation mixture was transformed in Escherichia coli DH5α chemocompetent cells (Chung et al., 1989) for plasmid DNA propagation and sequencing (pET30a-HIS-AgLTP24, supplemental Fig. S1). Plasmids DNA was prepared using (Nucleospin Plasmid DNA kit Macherey Nagel) from 4 independent clones, 2 ml of each E. coli culture grown overnight in 5ml LB medium at 37°C with shaking. Cloned insert DNA was checked by Sanger sequencing (Biofidal, Lyon, France) and used for electrotransformation into iSHuffle T7 lysY E. coli cells (NEB, Evry, France), a strain used to obtain proteins with disulphide bonds (Lobstein et al., 2012) .
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