We inserted the full-length OSMYB5P cDNA from rice into the pGEX-2T vector (Amersham, Buckinghamshire, UK) using XhoI restriction sites at the 5′ and 3′ ends of the cDNA fragment (S3 Table ). The pGEX-2T:OsMYB5P clone was introduced into the BL21 DE3 strain (Merck KGaA, Darmstadt, Germany) of E. coli. Expression of OSMYB5P protein was induced by applying 0.5 mM IPTG for 3 h at 30 °C. The recombinant OSMYB5P-GST protein extracts were purified by affinity chromatography using glutathione-agarose resin (Amersham, Buckinghamshire, UK) according to the manufacturer’s recommendations.
Bl21 de3 strain
Manufactured by Merck Group
Sourced in Germany
The BL21(DE3) strain is a common bacterial expression strain used in molecular biology laboratories. It is derived from the Escherichia coli B strain and is designed for the efficient expression of recombinant proteins. The strain contains the T7 RNA polymerase gene under the control of the lac operon, allowing for inducible expression of target genes.
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3 protocols using bl21 de3 strain
1
Recombinant Expression of OsMYB5P Protein
2
Expression and Purification of NHERF Proteins
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MBP–NHERF1/2 and GST-N–ezrin (GST-tagged N-terminal ezrin 1–309 aa) were purified as previously described [7 (link),33 (link)]. DNAs encoding the C-terminal fragments of NHERF1 and NHERF2 were generated by PCR and inserted into pET32a (EMD Millipore) for expressing both His6 and thioredoxin (His6–thioredoxin) fused recombinant proteins (Figure 1 ). These constructs were transformed into BL21(DE3) strain (EMD Millipore) for IPTG-induced protein expression. Proteins were purified with Ni-NTA resin following the manufacturer's instructions. Purified proteins were supplemented with 10% glycerol and 10 mM DTT and stored at −80°C.
3
Purification of Rabbit NHE3 Fragment
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cDNA fragments encoding rabbit NHE3 carboxyl-terminus fragment 642C (amino acids P642–M832) were amplified by PCR and inserted into the previously constructed vector pMBP (Yang et al., 2014 (link)) between BamHI and XhoI.
Expression constructs were transformed into BL21(DE3) strain (EMD Millipore, Billerica, MA). When the bacterial culture reached an OD of ∼0.8, protein expression was induced with 0.3 mM isopropyl-β-d -thiogalactoside at 16°C overnight. MBP-tagged proteins were purified with a column packed with amylose resin by gravity flow following the New England Biolabs manuals of the pMAL system. GST-tagged protein was purified in a gravity-flow column following the instructions from GE Healthcare. Purified proteins were concentrated by Amicon Ultra-15 Centrifugal filter units (EMD Millipore), supplemented with 10% glycerol and 10 mM dithiothreitol, and stored at −80oC.
Expression constructs were transformed into BL21(DE3) strain (EMD Millipore, Billerica, MA). When the bacterial culture reached an OD of ∼0.8, protein expression was induced with 0.3 mM isopropyl-β-
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