P. gingivalis apoHmuY lacking the first 25 residues (NCBI accession number CAM 31898) was expressed using a pHmuY11 plasmid and Escherichia coli ER2566 cells (New England Biolabs) and purified from a soluble fraction of the E. coli cell lysate as described previously [25 ]. As the soluble protein released from the cell membrane, the purified HmuY lacked the signal peptide and first five amino acid residues (CGKKK) of the nascent secreted protein [25 ,27 (link)].
Escherichia coli er2566 cells
Manufactured by New England Biolabs
Escherichia coli ER2566 cells are a laboratory strain of the bacterium Escherichia coli. They are commonly used as a host strain for the expression and purification of recombinant proteins.
Automatically generated - may contain errors
Lab products found in correlation
Superdex 200 10 300 column, by GE Healthcare (1 mentions)
Kta explorer system, by GE Healthcare (1 mentions)
Vivaspin concentrator, by GE Healthcare (1 mentions)
His select nickel affinity gel, by Merck Group (1 mentions)
Superdex 75 10 300, by GE Healthcare (1 mentions)
Chitin affinity chromatography, by New England Biolabs (1 mentions)
Ptyb1 vector, by New England Biolabs (1 mentions)
Superdex 75, by GE Healthcare (1 mentions)
5 protocols using escherichia coli er2566 cells
1
Purification of P. gingivalis apoHmuY
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P. gingivalis apoHmuY lacking the first 25 residues (NCBI accession number CAM 31898) was expressed using a pHmuY11 plasmid and Escherichia coli ER2566 cells (New England Biolabs) and purified from a soluble fraction of the E. coli cell lysate as described previously [25 ]. As the soluble protein released from the cell membrane, the purified HmuY lacked the signal peptide and first five amino acid residues (CGKKK) of the nascent secreted protein [25 ,27 (link)].
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P. gingivalis apoHmuY lacking the first 25 residues (NCBI accession number CAM 31898) was expressed using a pHmuY11 plasmid and Escherichia coli ER2566 cells (New England Biolabs) and purified from a soluble fraction of the E. coli cell lysate as described previously [25 ]. As the soluble protein released from the cell membrane, the purified HmuY lacked the signal peptide and first five amino acid residues (CGKKK) of the nascent secreted protein [25 ,27 (link)].
2
Mutagenesis of P. gingivalis HmuY Protein
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The DNA sequence encoding P. gingivalis HmuY polypeptide part lacking the first 25 residues (NCBI accession no. CAM 31898) and cloned into pHmuY11 plasmid [15 (link)] served as a template for mutagenesis. Single tryptophan residues were replaced by alanine or tyrosine residues using QuikChange II XL Site-Directed Mutagenesis Kit (Stratagene). Primers designed and used in this study are listed in Table 1 . All HmuY protein variants were overexpressed using respective plasmids and Escherichia coli ER2566 cells (New England Biolabs), and purified from a soluble fraction of E. coli lysates as previously described [16 (link)]. Molecular modeling of HmuY protein variants was performed with the GROMOS96 implementation of Swiss-PdbViewer (version 4.1).
3
Purification and Labeling of FtsZ, sZipA, and DynA
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FtsZ
was overexpressed in Escherichia coli ER2566 cells (New England Biolabs), purified as described earlier,44 (link) and stored in storage buffer (50 mM Tris pH7.5,
500 mM KCl, 5 mM MgCl2, 10% glycerol). Labeling of FtsZ
with Alexa Fluor 488 NHS ester was performed as described previously.45 (link) Purified sZipA was kindly provided by Dr. G.
Rivas.46 (link)Bacillus subtilis DynA was overexpressed from pET16b (kindly provided by Dr. M. Bramkamp)
and purified essentially as described (Bürmann et al. 2011)
but eluted with a linear imidazole gradient instead of a step elution.
A solution of ∼10 μM Dynamin was labeled with 8-fold
molar excess of Alexa Fluor 488 maleimide in the presence of 0.05
mM TCEP (45 min at room temperature), quenched with 10 mM β-mercaptoethanol,
and separated from free label on a Superdex S200 column equilibrated
with T5 buffer (50 mM Tris/HCl pH8.0, 500 mM NaCl, 10% glycerol).
was overexpressed in Escherichia coli ER2566 cells (New England Biolabs), purified as described earlier,44 (link) and stored in storage buffer (50 mM Tris pH7.5,
500 mM KCl, 5 mM MgCl2, 10% glycerol). Labeling of FtsZ
with Alexa Fluor 488 NHS ester was performed as described previously.45 (link) Purified sZipA was kindly provided by Dr. G.
Rivas.46 (link)Bacillus subtilis DynA was overexpressed from pET16b (kindly provided by Dr. M. Bramkamp)
and purified essentially as described (Bürmann et al. 2011)
but eluted with a linear imidazole gradient instead of a step elution.
A solution of ∼10 μM Dynamin was labeled with 8-fold
molar excess of Alexa Fluor 488 maleimide in the presence of 0.05
mM TCEP (45 min at room temperature), quenched with 10 mM β-mercaptoethanol,
and separated from free label on a Superdex S200 column equilibrated
with T5 buffer (50 mM Tris/HCl pH8.0, 500 mM NaCl, 10% glycerol).
4
Purification and Characterization of Mouse TACO1
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The coding sequence of mouse TACO1, lacking its N-terminal mitochondria targeting sequence, (amino acids 27–294, NP_081622.1) was cloned into pETM30 and expressed as a fusion to an N-terminal His tag and glutathione S-transferase in Escherichia coli ER2566 cells (New England Biolabs). Wild-type and mutant proteins (Mutagenex) were purified using HIS-Select Nickel Affinity Gel according to the manufacturer's instructions (Sigma-Aldrich). The eluted protein was dialysed against 25 mM Tris-HCl (pH 7.4), 0.2 M NaCl, 0.5 mM EDTA, 2 mM DTT, 10% glycerol and further purified by gel filtration using an ÄKTAexplorer system (GE) with a Superdex 200 10/300 column with a total bed volume of 120 ml and concentrated using Vivaspin concentrators (GE) before use in RNA-binding assays. Protein concentration was determined by the bicichroninic acid assay using BSA as a standard.
Protein used for crystallization was purified as above with the following modifications: During dialysis in H-0.05, the fusion tag was removed by Tobacco Etch Virus at a protease/protein molar ratio of 1:50 followed by purification on a heparin column equilibrated in H-0.05 and eluted with H-0.3 prior to gel filtration.
Protein used for crystallization was purified as above with the following modifications: During dialysis in H-0.05, the fusion tag was removed by Tobacco Etch Virus at a protease/protein molar ratio of 1:50 followed by purification on a heparin column equilibrated in H-0.05 and eluted with H-0.3 prior to gel filtration.
5
Recombinant BAX Mutant Purification
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Recombinant Proteins BAX mutants were generated by site-directed mutagenesis of BAX constructs from Homo sapiens, Mus musculus or Ictalurus punctatus. BAX constructs have their wild-type cysteine residues mutated into serine (C62S, C126S for hsBAX and mmBAX, and C51S, C117S for ipBAX). hsBAXDC26 and hsBAX P168G have two C-terminal serine residues due to the cloning strategy. BAX constructs were cloned into pTYB1 vector (New England Biolabs). Recombinant BAX constructs were expressed and purified according to a described protocol (Suzuki et al., 2000) . Proteins were expressed in Escherichia coli ER2566 cells (New England Biolabs), lysed in TBS (20 mM Tris pH 8, 150 mM NaCl) and purified in two steps: chitin affinity chromatography (New England Biolabs) followed by size exclusion chromatography (Superdex75, GE Healthcare Life Sciences) in TBS. For analytical SEC, proteins concentrated at 10 mg/mL were loaded on Superdex75 10/300 (GE healthcare Life Science) and eluted in TBS supplemented with 2 mM dithiothreitol.
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