Cloning and Purification of NtCDPK1 Variants

Plasmids pET16b-NtCDPK1 wild-type (WT), pGEX-4T-1-NtCDPK1 WT, S6A, T21A, S6A/T21A, D219N, and S6A/T21A/D219N, and pMALc2-RSG were previously generated [17 (link),29 (link)]. NtCDPK1 S6A/D219N and T21A/D219N were generated using overlap extension PCR and seamless ligation cloning extract (SLiCE) [30 (link)] with a plasmid harbouring full-length NtCDPK1 as a template and appropriate primers (S1 Table). PCR products were cloned into pGEX-4T-1 (GE Healthcare). Glutathione S-transferase (GST)-NtCDPK1s, maltose-binding protein (MBP)-RSG, and 10×His-tagged (His)-NtCDPK1 were expressed in Escherichia coli harbouring pGEX-4T-1-NtCDPK1, pMALc2-RSG, or pET16b-NtCDPK1, respectively, and purified by glutathione-Sepharose 4B (GE Healthcare), Amylose Resin (New England Biolabs), or COSMOGEL His-Accept (Nacalai Tesque), respectively.

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Ito T., Ishida S, & Takahashi Y. (2018). Autophosphorylation of Ser-6 via an intermolecular mechanism is important for the rapid reduction of NtCDPK1 kinase activity for substrate RSG. PLoS ONE, 13(4), e0196357.

Publication 2018

pGEX-4T-1 glutathione-Sepharose 4B Amylose Resin COSMOGEL His-Accept

Amylose Escherichia coli Glutathione Glutathione s transferase Ligation Maltose binding protein Plasmid Primers Resin Sepharose 4b

Corresponding Organization : Hiroshima University

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Variable analysis

independent variables

Plasmid type (pET16b-NtCDPK1 wild-type (WT), pGEX-4T-1-NtCDPK1 WT, S6A, T21A, S6A/T21A, D219N, and S6A/T21A/D219N, and pMALc2-RSG)

dependent variables

Expression and purification of Glutathione S-transferase (GST)-NtCDPK1s, maltose-binding protein (MBP)-RSG, and 10×His-tagged (His)-NtCDPK1

control variables

Plasmid harbouring full-length NtCDPK1 as a template for PCR
Appropriate primers for generating NtCDPK1 S6A/D219N and T21A/D219N
Expression host Escherichia coli

positive controls

Not explicitly mentioned

negative controls

Not explicitly mentioned

Annotations

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