The miRFP670 and miRFP709 genes were cloned into a pBAD/His-B vector (Invitrogen). Site-specific mutagenesis of miRFP670 was performed using QuikChange kit (Stratagene), resulting in miRFP670/C20A mutant. The miRFP670, miRFP670/C20A and miRFP709 proteins with polyhistidine tags on the N-termini were expressed in LMG194 bacterial cells (Invitrogen) bearing the pWA23h plasmid encoding a heme oxygenase under the rhamnose promoter5 (link). To initiate protein expression, the bacterial cells were grown in RM medium supplemented with ampicillin, kanamycin and 0.02% rhamnose for 5 h at 37 °C. Then 0.002% arabinose was added, and the bacterial culture was incubated for an additional 12 h at 37 °C followed by 24 h at 18 °C. The resulting holoproteins were purified using a Ni-NTA agarose (Qiagen). An Ni-NTA elution buffer contained 100 mM EDTA and no imidazole. After elution, the buffer was substituted with phosphate-buffered saline using PD-10 desalting columns (GE Healthcare). In experiment with apoproteins, they were purified without co-expression of the heme oxygenase and then dissolved in 20 mM Tris-HCl buffer with 150 mM NaCl at pH or pD 8.0. The protein concentrations were ~0.45 and ~0.9 mM for time-resolved absorption and Raman experiments, respectively.
Lmg194 bacterial cells
Manufactured by Thermo Fisher Scientific
The LMG194 bacterial cells are a laboratory strain commonly used for research and experimental purposes. These cells are designed to serve as a host for various molecular biology applications. Their core function is to provide a well-characterized and reliable platform for gene expression, protein production, and other related studies. The specific details and intended use of these cells may vary depending on the research context and requirements.
Automatically generated - may contain errors
6 protocols using lmg194 bacterial cells
1
Purification and Characterization of miRFP Proteins
2
Purification of Phytochrome Photoreceptors
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BphP1 and BphP1-mRuby2 proteins with polyhistidine tags on the N-terminus were expressed in LMG194 bacterial cells (Life Technologies-Invitrogen) containing a pWA23h plasmid encoding heme oxygenase for biliverdin synthesis in Escherichia coli18 (link). The bacterial cells were grown in RM medium supplemented with ampicillin, kanamycin and 0.02% rhamnose for 6-8 h followed by an induction of the protein expression by adding of 0.002% arabinose. The proteins were purified using a Ni-NTA agarose (Qiagen). The PpsR2 and PpsR2-mVenus proteins with a Strep-tag-II at the N-terminus and a polyhistidine tag at the C-terminus was expressed in BL21(DE3) bacterial cells grown in LB medium supplemented with ampicillin for 6 h, followed by an induction of a protein expression with 250 μM IPTG. The proteins were first purified with a Ni-NTA agarose (Qiagen) followed by purification with a Strep-Tactin sepharose (IBA Lifesciences).
3
Purification of iRFP Fluorescent Proteins
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The iRFP702, iRFP713 and iRFP720 genes were cloned in the pBAD/His-B vector (Invitrogen)14 (link). The iRFP702, iRFP713 and iRFP720 proteins with polyhistidine tags on the N-termini were expressed in LMG194 bacterial cells (Invitrogen) bearing the pWA23h plasmid encoding heme oxygenase under the rhamnose promoter13 14 (link). To initiate protein expression, bacterial cells were grown in RM medium supplemented with ampicillin, kanamycin and 0.02% rhamnose for 5 h at 37 °C. Then 0.002% arabinose was added and bacterial culture was incubated for additional 12 h at 37 °C following by 24 h at 18 °C. Proteins were purified using Ni-NTA agarose (Qiagen). Ni-NTA elution buffer contained no imidazole and 100 mM EDTA. The elution buffer was substituted with PBS buffer using PD-10 desalting columns (GE Healthcare).
4
Bacterial Expression and Purification of Near-Infrared Fluorescent Proteins
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The BphP1-FP, iRFP670 and iRFP682 genes were cloned into the pBAD/His-B vector (Invitrogen). Site-specific mutagenesis was performed using QuikChange kit (Stratagene). The proteins with polyhistidine tags on the N-termini were expressed in LMG194 bacterial cells (Invitrogen) bearing the pWA23h plasmid encoding heme oxygenase under the rhamnose promoter14 (link). To initiate protein expression, bacterial cells were grown in RM medium supplemented with ampicillin, kanamycin and 0.02% rhamnose for 5 h at 37 °C. Then 0.002% arabinose was added and bacterial culture was incubated for additional 12 h at 37 °C followed by 24 h at 18 °C. Proteins were purified using Ni-NTA agarose (Qiagen). Ni-NTA elution buffer contained no imidazole and 100 mM EDTA. The elution buffer was substituted with PBS buffer using PD-10 desalting columns (GE Healthcare). For spectroscopic experiments, the samples were diluted in PBS buffer composed of 137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4 and 2 mM KH2PO4 at pH 7.4.
5
Production and Purification of miRFP Proteins
6
Purification of Phytochrome Photoreceptors
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