The dual expression plasmid was transformed into Rosetta II (DE3) (Invitrogen) E. coli strain, grown in Luria Bertani medium, and induced with 1 mM isopropyl-β-d-thiogalactopyranoside at an A600 ~ 0.8 for 2 hours. The bacterial pellets from 1L of growth media were resuspended in 30 mL of B-PER reagent (Thermo Scientific) and expressed protein purified from the lysis supernatant by Nickel affinity and gel filtration (Superdex 75 26/60, GE Healthcare) chromatography. The purified protein was concentrated and passed over the gel filtration column a second time before Western blot analysis.
Rosetta 2 de3
Manufactured by Thermo Fisher Scientific
Sourced in France, United States
The Rosetta II (DE3) is a bacterial expression strain developed by Merck KGaA, Darmstadt, Germany. It is designed to enhance the expression of proteins that contain rare codons found in the E. coli genome. The strain is derived from the BL21 (DE3) expression host and contains the pRARE plasmid, which provides tRNAs for the rare codons AUA, AGG, AGA, CUA, CCC, and GGA.
Automatically generated - may contain errors
Lab products found in correlation
B per reagent, by Thermo Fisher Scientific (2 mentions)
Superdex 75 26 60, by GE Healthcare (1 mentions)
Magicmedia, by Thermo Fisher Scientific (1 mentions)
Bradford reagent, by Bio-Rad (1 mentions)
Ni2 nta resin, by Qiagen (1 mentions)
E coli bl21 de3, by Thermo Fisher Scientific (1 mentions)
Ni nta agarose bead, by Qiagen (1 mentions)
Restriction enzyme, by New England Biolabs (1 mentions)
Isoliquiritigenin, by Merck Group (1 mentions)
Naringenin chalcone, by Merck Group (1 mentions)
Bacto agar, by BD (1 mentions)
Bacto tryptone, by BD (1 mentions)
8 protocols using rosetta 2 de3
1
Recombinant Protein Purification in E. coli
2
Co-expression and Purification of PACRG and DNALI1
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Full-length mouse Pacrg cDNA (amplified by a forward primer: 5’-GAATTC GGTGCCGCGCGGCAGCATGCCGAAGAGGACTAAACTG-3’ and a reverse primer: 5’-GTCGAC TCAGTTCAGCAAGCACGACTC-3’ ) was cloned into the upstream multiple clone site of the dual expression vector pCDFDuet-1 to create the PACRG/pCDFDuet-1 plasmid, and the translated protein was tagged with hexahistidine. The full-length mouse Dnali1 cDNA (amplified by a forward primer: 5’-GATATC GATGATACCCCCAGCAGACTCTC-3’ and a reverse primer: 5’-CTCGAG TCACTTCTTCGGTGCGATAATG-3’ ) was inserted into the downstream multiple cloning site to create the PACRG/DNALI1/pCDFDuet-1 plasmid. The dual expression plasmid was transformed into the Rosetta II (DE3) (Invitrogen) Escherichia coli strain, grown in Luria Bertani medium, and induced with 1 mM isopropyl-β-d-thiogalactopyranoside at an A600 ~0.8 for 2 hr. The bacterial pellets from 1 L of growth media were re-suspended in 30 mL of B-PER reagent (Thermo Scientific, Waltham, MA, USA), and co-expressed proteins were purified from the lysis supernatant by nickel affinity chromatography. The purified protein was analyzed by western blotting.
3
Purification of Recombinant APE1 Protein
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To purify APE1 expressed as a recombinant protein, 1 L of culture (in LB broth) of Escherichia coli strain Rosetta II(DE3) (Invitrogen, France) carrying the pET11a-APE1 construct was grown with 50 mg mL À1 ampicillin at 37 1C until absorbance at 600 nm (A 600 ) reached 0.6-0.7; APE1 expression was induced overnight with 0.2 mM isopropyl-b-D-thiogalactopyranoside. The method for isolation of wild-type APE1 was described previously. 29, 31 The protein concentration was measured by the Bradford method; 32 the stock solution was stored at À20 1C.
4
Expression and Purification of Kinases
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Kinase screen (Addgene kit #1000000094) was received as bacterial glycerol stocks. Expression and purification protocols were adapted from the published protocol in Albanese et al. (2018) (link). Plasmids containing all the Ser/Thr kinases were isolated by miniprep of overnight cultures inoculated from glycerol stocks. Plasmids containing kinases conferred carbenicillin resistance and were cotransformed into Rosetta2 DE3 cells (Novagen) with a lambda phosphatase-encoding plasmid conferring resistance for spectinomycin (provided in the Addgene kit). 60 ml cultures containing MagicMedia (Invitrogen), 100 µg/ml carbenicillin, and 100 µg/ml spectinomycin were inoculated with Rosetta2 DE3 cotransformations and grown at 37°C for 4 h, then at 16°C for 40 h. Cells were harvested by centrifugation at 3,000 ×g, resuspended in buffer A, and frozen in liquid N2. Frozen cell pellets were thawed to room temperature followed by sonication on ice until lysates were homogenous. Lysates were cleared by centrifugation at 3,000 ×g at 4°C, then loaded onto 100 µl Ni2+-NTA resin (Qiagen) preequilibrated with buffer A. Resin was washed with 100 CV buffer A, then eluted into 250 µl buffer B. Protein concentration was measured by Bradford reagent (BioRad), then aliquoted and frozen in liquid N2 and stored at −80°C until needed.
5
Purification of Snl1 Protein from E. coli
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The plasmid pPROEX-HTb-SNL1-S was transformed into E. coli strain Rosetta 2(DE3) (Invitrogen). The cells were grown at 37° until optical density at 600nm (OD) reaches 0.5−0.6. Culture was induced with 0.3 mM IPTG and further grown at 20° overnight. Cells were harvested and lysed in lysis buffer (25 mM HEPES, 150 mM NaCl, protease cocktail inhibitor) using lysozyme followed by sonication. Lysate was spun at 10,000 g for 45 min, and supernatant was loaded onto cobalt-based Talon metal affinity resin. Unbound protein was washed, and Snl1 was eluted with 300 mM imidazole and subsequently dialyzed using storage buffer (25 mM HEPES, 150 mM NaCl). Protein purity was verified using sodium dodecyl sulfate polyacrylamide gel electrophoresis and identity was confirmed using western blot analysis with anti-His tag antibody. Proteins were purified to near homogeneity (Supporting Information , Figure S1 ). If required to improve purity, protein was further purified by Superdex 75 gel-filteration chromatography.
6
Purification of Cpr7 and Ure2 Proteins
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For Cpr7 purification, the plasmid pET29bHTV-CPR7 was transformed into Escherichia coli strain Rosetta 2(DE3) (Invitrogen). The pre-grown culture of optical density at 600nm (O.D.600nm) ~0.6 was induced with isopropyl-β-D-thiogalactopyranoside (IPTG) at 18°C. Cells were lysed and the protein was purified from supernatant using cobalt based Talon metal affinity resin. Cpr7 was eluted with 300mM imidazole and further incubated overnight with His6-TEV (molar ratio, Cpr7/His-TEV:20/1) protease at 4°C. The sample was extensively dialyzed and further incubated with cobalt metal affinity column and the His6-tag cleaved Cpr7 protein was collected as unbound fraction. The protein was further purified using anion exchange (Mini Q) chromatography. Protein purity was verified using SDS-PAGE and identity was confirmed using Mass spectrometry. Cpr6 and 7TPR were purified using the procedure described above for Cpr7.
The plasmid pKT41-Ure2 (kind gift from Dr. Reed Wickner, National Institute of Health, Bethesda) encodes Ure2 with an N-terminal Hexa-His-tag. The plasmid was transformed into E.coli strain Rosetta 2(DE3) and Ure2p was purified using cobalt based Talon metal affinity resin as described before [52 (link)].
The plasmid pKT41-Ure2 (kind gift from Dr. Reed Wickner, National Institute of Health, Bethesda) encodes Ure2 with an N-terminal Hexa-His-tag. The plasmid was transformed into E.coli strain Rosetta 2(DE3) and Ure2p was purified using cobalt based Talon metal affinity resin as described before [52 (link)].
7
Rice Developmental and Stress Profiling
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Wild-type rice (Oryza sativa L. subsp. Japonica cv. Dongjin) seeds were sterilized with 50% bleach for 30 min and germinated on Murachige and Skoog medium (Duchefa Biochemie, Haarlem, The Netherlands) in a growth chamber with a 16 h light/8 h dark photoperiod at 28 °C for two weeks. The rice seedlings were transferred to soil and grown in a greenhouse at 28 °C during the day. Shoot and root samples were collected from two-week-old rice seedlings. Leaves, stems, and roots were obtained from eight-week-old rice plants during the vegetative growth period. Panicles were harvested from 14-week-old rice plants. Eight-week-old rice plants were irradiated with UV light to examine the UV stress response. UV treatment and HPLC analysis were carried out according to the methods described by Park et al. [25 (link)].
Naringenin chalcone and isoliquiritigenin were purchased from Sigma-Aldrich (St. Louis, MO, USA). E. coli BL21(DE3) and Rosetta 2(DE3) strains were obtained from Thermo Fisher Scientific (Waltham, MA, USA). Ni-NTA Agarose beads and CM Sepharose Fast Flow resin were purchased from Qiagen (Hilden, Germany) and Cytiva (Marlborough, MA, USA), respectively. Restriction enzymes were bought from New England Biolabs (Ipswich, MA, USA) and Enzynomics (Daejeon, Korea). Reagents for buffers and media were obtained from Sigma-Aldrich and Duchefa Biochemie.
Naringenin chalcone and isoliquiritigenin were purchased from Sigma-Aldrich (St. Louis, MO, USA). E. coli BL21(DE3) and Rosetta 2(DE3) strains were obtained from Thermo Fisher Scientific (Waltham, MA, USA). Ni-NTA Agarose beads and CM Sepharose Fast Flow resin were purchased from Qiagen (Hilden, Germany) and Cytiva (Marlborough, MA, USA), respectively. Restriction enzymes were bought from New England Biolabs (Ipswich, MA, USA) and Enzynomics (Daejeon, Korea). Reagents for buffers and media were obtained from Sigma-Aldrich and Duchefa Biochemie.
8
Recombinant Protein Expression in E. coli
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C41(DE3) and BL21(DE3) E. coli strains were purchased from MilliporeSigma. C41pLysS, C43pLysS, and Rosetta 2(DE3) E. coli strains were procured from Thermo scientific. Bacto tryptone, Bacto yeast, and Bacto Agar were purchased from BD Biosciences. Anti-His tag Ab (Clone 27E9, Cat. No. 2366) and anti-EGFR mAb (Clone D38B1, Cat. No. 4267) were purchased from Cell Signaling Technology, while anti-Ezrin Ab (Clone H-276, Cat No. SC-20773) was bought from Santa Cruz Biotechnology. HisTrap HP affinity Ni-NTA column was purchased from GE healthcare. Anti-His tag Ab Magnetic bead conjugate (Cat. No. 8811) and Protein A/G agarose beads (Cat. No. 9863) were purchased from Cell Signaling Technology. Anti-MUC4β mAb (Clone 6E8) was generated using recombinant rMUC4β protein as an immunogen. More details on the construction of recombinant pET-28a-MUC4β are mentioned in supporting information.
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