Primers were designed using the full-length gene sequence of A. castellanii Neff inosineuridine preferring nucleotide hydrolase (IPNH) provided by the NCBI database (GenBank accession no: XP_004334078). The coding sequence of A. castellanii (ATCC 30868) IPNH was amplified by polymerase chain reaction using the following primers: 5’-ATGACCGCGAGCAAGTCGTT-3’ (forward primer), and 5’-CTACCAGCGGCGTCGCCTGT-3’ (reverse primer). Escherichia coli BL21 (DE3) was used as an expression host of pGEX 4T-3 GST expression vector (GE Healthcare, Buckinghamshire, UK). Isopropyl-β-D-thiogalactopyranoside (IPTG) was added to a final concentration of 1 mM for expression of GST-IPNH fusion protein in E. coli, and incubated for 4 h. Protein expressions were confirmed using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and Coomasie blue staining (Elpisbio, Daejeon, Korea). Target protein was extracted using the EzWay™ PAG Protein Elution Kit V2 (KOMABIOTECH, Seoul, Korea). Protein samples were concentrated by Amicon Ultra-4 centrifugal filter device (Merck Millipore, Burlington, MA, USA).
Pgex 4t 3 gst expression vector
Manufactured by GE Healthcare
Sourced in United Kingdom, Germany
The PGEX-4T-3 GST expression vector is a tool used for the production and purification of recombinant proteins in bacterial systems. It contains the glutathione S-transferase (GST) gene, which allows for the efficient capture and isolation of the target protein. The vector supports high-level expression of fusion proteins and provides a simple and effective method for the purification of these proteins.
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Lab products found in correlation
3 protocols using pgex 4t 3 gst expression vector
1
Recombinant Expression of A. castellanii IPNH
2
Cloning and Sequencing of Rtn4b Motifs
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The rtn4b-M1-M4 region (Shypitsyna et al., 2011) (link) was amplified by PCR from a TOPO2.1 vector containing the rtn4b ORF (Pinzon-Olejua et al., 2014) (link). Forward (FW) 5 0 -GGGAATTCT-AGCCCGTCTCCAGACCTGCTC-CAGGA-3 0 and reverse (RV) 5 0 -GGGTCGACCTA-CTGCAGACCCTGGAG-CAGCTCTGCC-3 0 primers containing EcoRI and SalI restriction sites were designed to amplify 490 base pairs including the M1 to M4 motifs. The PCR product was digested with EcoRI and SalI and cloned in frame after the thrombin cleavage site of the pGEX-4T-3 GST expression vector (GE Healthcare, Braunschweig, Germany). All the above-mentioned PCR reactions were performed with the Phusion high-fidelity DNA polymerase (Finnzymes/Thermo Fisher) and positive clones were further sequenced.
3
Recombinant Rat M1-4 Protein Expression
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The pET28 expression vector containing the Rat-NIGD20 construct was kindly provided by M. E. Schwab (University and ETH Z€ urich, Switzerland) . The delta20 sequence was PCR-amplified from this vector and inserted into the pGEX-KG plasmid.
To create the recombinant Rat M1-4 protein, the original pGEX-D20 plasmid was used as template for elongation by PCR with FW primer 5 0 -AAAGGATCCACGCCAGATTTAGTTCAGGAAGCATGTGAAAGTGAA-CTGAATGAAGCCACAGGTACAAAGATTGCTTATGAAACAAAAGTGG-3 0 and RV primer 5 0 -GAGAGCTTTGTTTCTTTAATTAAATCACACG-CAATGGATA TATAAGGAG-3 0 containing BamHI and PacI restriction sites. PCR product was digested with the respective enzymes and cloned in frame behind the thrombin cleavage site of the pGEX-4T-3 GST expression vector (GE Healthcare).
To create the recombinant Rat M1-4 protein, the original pGEX-D20 plasmid was used as template for elongation by PCR with FW primer 5 0 -AAAGGATCCACGCCAGATTTAGTTCAGGAAGCATGTGAAAGTGAA-CTGAATGAAGCCACAGGTACAAAGATTGCTTATGAAACAAAAGTGG-3 0 and RV primer 5 0 -GAGAGCTTTGTTTCTTTAATTAAATCACACG-CAATGGATA TATAAGGAG-3 0 containing BamHI and PacI restriction sites. PCR product was digested with the respective enzymes and cloned in frame behind the thrombin cleavage site of the pGEX-4T-3 GST expression vector (GE Healthcare).
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