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Pet28a

Manufactured by Agilent Technologies

The PET28a is a plasmid vector used for the expression of recombinant proteins in Escherichia coli. It contains a T7 promoter and a His-tag sequence for efficient purification of the expressed proteins.

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3 protocols using pet28a

1

Cloning and Mutagenesis of Tomato Chlorosis Virus p22 Gene

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For gene expression, the ToCV p22 gene was PCR-amplified from plasmid pGEM-T-p22 (Cañizares et al., 2008 (link)) using the Expand High Fidelity PCR system (Roche Diagnostics) and primers MA 1287 and MA 1288 with specific restriction sites. The PCR product was digested, purified and cloned into Escherichia coli expression vector pET-28a(+) (Novagen), resulting in the pET28a-p22 construct, harboring the p22 gene fused to a sequence coding for a hexa-His tag. To generate the p22Δ2Cys construct, which lacked four amino acid residues including two cysteines located between positions 39–42 (C-terminal region), amplification from plasmids pET28a-p22 and pBin35S-p22 (Cañizares et al., 2008 (link)) was performed. Mutations were introduced by PCR using the QuikChange II XL Site-Directed Mutagenesis Kit (Agilent Technologies) and the specific primers MA 1616 and MA 1617, generating pET28a-p22Δ2Cys and pBIN35S-p22Δ2Cys.
The constructs pBIN35S-p22 and pETDuet.SAHH have been previously described (Cañizares et al., 2008 (link), Cañizares et al., 2013 (link)). The 35S-dsSAHH construct was kindly provided by Dr. David M. Bisaro (The Ohio State University, Columbus, OH, USA), and plasmids 35S-GFP and 35S-p19 were kindly provided by Dr. David C. Baulcombe (University of Cambridge, Cambridge, United Kingdom).
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2

Recombinant Expression of Arabidopsis TOP1 and TOP2

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The cDNA sequences encoding A. thaliana TOP1 (AT5G65620) and TOP2 (AT5G10540) were cloned into pET-28a (Agilent) in a translational frame with 6xHis N-terminal tags. Cysteine mutagenesis was performed by site-directed mutagenesis, using the wild-type cDNAs, gene-specific primers, and Phusion High-Fidelity DNA Polymerase (New England Biolabs). The PCR products were separated on agarose gels (0.8%), purified using the QIAquick gel extraction Kit (Qiagen), and cloned into pET-28a using NheI and SalI restriction enzymes (NEB) and T4 DNA ligase (NEB). The plasmids were used for the expression of TOPs in E. coli. Additional details are described in Supplemental Methods.
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3

Purification of Dmr1-MBP-His6 Fusion Protein

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Cloning, expression, and purification of the Dmr1-MBP-His6 fusion protein was preformed essentially as described previously (Puchta et al. 2010 (link)). For purification of the Dmr1-His6 fusion protein, the codon-optimized (for yeast) sequence of DMR1 ORF (without the 26 amino acid amino-terminal mitochondrial targeting sequence) was obtained by custom gene synthesis (Generay Biotech Co.) and cloned in the pET28a vector (Novagen). The pET28a::DMR1 vector was transformed into BL21(DE3) CodonPlus-RIL E. coli (Agilent). An amount of 1 mL of the overnight culture was inoculated into 500 mL of AIM (auto induction medium, Formedium), and the cells were grown at 23°C for 36 h. Metal affinity and size exclusion protein purification procedures were performed as described previously (Malecki et al. 2008 (link)). The MBP-His6 protein used as the negative control was expressed from the pMM41 plasmid (Mayekar et al. 2013 (link)) and purified using the same protocol. Purity and composition of the protein preparations was assessed using LC–MS–MS/MS mass spectrometry on the Orbitrap (Thermo Fisher) spectrometer at the Laboratory of Mass Spectrometry, IBB PAS. The purified proteins were stored in a buffer containing 0.5 M NaCl, 50 mM Tris-HCl pH 8, 20% glycerol.
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