Phmgwa vectors

Manufactured by Addgene

PHMGWA vectors are plasmid-based expression vectors designed for recombinant protein production. These vectors contain a polyhistidine tag (His-tag) sequence for affinity purification, as well as a tobacco etch virus (TEV) protease cleavage site to facilitate tag removal. The vectors support inducible expression of the target protein in a variety of host organisms.

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Lab products found in correlation

Synthetic dna, by Thermo Fisher Scientific (1 mentions) Ni sepharose column, by GE Healthcare (1 mentions) Edta free antiprotease, by Roche (1 mentions)

2 protocols using phmgwa vectors

Cloning and Purification of ARF and RAV Proteins

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cDNA sequences coding for potential ancestors and the corresponding mutants were constructed as synthetic DNA (Thermofisher). KnRAV and CaARF (full-length, fragments (CaARF-DBD (residues 1–421), CaARF-PB1 (residues 644–750), KnRAV-DBD (residues 256–523), KnRAV-PB1 (residues 724–798)) or mutants) coding sequences were cloned into pETM40 plasmid (EMBL) that contains a MBP-tag in the N-terminal region except for PB1 domains from both proteins that were cloned into pETM11 (EMBL) that confers a N-terminal His-tag.
KnRAV and CaARF specific domains were isolated by PCR from synthetic cDNA sequences (S6 Table). Full-length ARF2, ARF5 and ARF10 were cloned into pHMGWA vectors (Addgene) containing N-terminal His-MBP-His tags.

Martin-Arevalillo R., Thévenon E., Jégu F., Vinos-Poyo T., Vernoux T., Parcy F, & Dumas R. (2019). Evolution of the Auxin Response Factors from charophyte ancestors. PLoS Genetics, 15(9), e1008400.

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Purification of ARF2 and ARF5 Proteins

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ARF2 and ARF5 coding sequences were cloned into pHMGWA vectors (Addgene) containing N-terminal His-MBP-His tags. His-MBP-Histagged ARF proteins were expressed in Escherichia coli Rosetta2 strain. Bacteria cultures were grown in liquid LB medium to an OD 600 nm of 0.6-0.9. Protein expression was induced with isopropyl-b-D-1thyogalactopiranoside (IPTG) at a final concentration of 400 mM. Protein production was done overnight at 18 C. Bacteria cultures were centrifuged, and the resulting pellets were resuspended in lysis buffer (Tris-HCl 20 mM [pH 8]; NaCl 500 mM; Tris(2-carboxyethyl) phosphine (TCEP) 1 mM for ARF2 and Tris-HCl 20 mM [pH 8]; NaCl 500 mM; EDTA 0.5 mM; PMSF 0.5 mM; TCEP 1 mM; Triton 0.2% (w/v) for ARF5) with EDTA-free antiprotease (Roche) for sonication. Proteins were separated from the soluble fraction on Ni-Sepharose columns (GE Healthcare) previously equilibrated with the corresponding lysis buffer. Elutions were done with Imidazole 300 mM diluted in the corresponding lysis buffer.

Stigliani A., Martin-Arevalillo R., Lucas J., Bessy A., Vinos-Poyo T., Mironova V., Vernoux T., Dumas R, & Parcy F. (2019). Capturing Auxin Response Factors Syntax Using DNA Binding Models. Molecular plant, 12(6).

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