Two DNA fragments each encoding a fluorescent protein fused to CBO1751 CBD were designed by inserting DNA encoding the amino acid residues 169–253 of CBO1751 (GenBank accession number CAL83288) to the 3′ end of the DNA encoding mCherry (AAV52164) or mTagBFP (AZQ25074), termed mCherry-CBD and mTagBFP-CBD, respectively. The DNA fragments were commercially synthesized and cloned in the NheI/SalI restriction sites of pET21b to generate a C-terminal fusion construct with a 6 × His tag (Biomatik Corporation, Ontario, Canada). The vectors were transformed into E. coli Rosetta 2 (DE3) pLysS cells (Merck Millipore). The expression of the His-tagged protein was induced with 1 mM IPTG at 30 °C for 8 h. The expressed protein was purified using metal-chelate affinity chromatography with Ni–IDA resin (Merck Millipore), as previously described [46 (link)]. The eluted proteins were dialyzed against Tris buffer (500 mM NaCl, 50% glycerol, 20 mM Tris–HCl, pH 7.9) for long-term storage or a PBS buffer (pH 7.4) for immediate use in coupling to magnetic beads.
E coli rosetta 2 de3 plyss cells
Manufactured by Merck Group
Sourced in Germany
E. coli Rosetta 2(DE3) pLysS cells are a competent bacterial strain commonly used in molecular biology research. They are designed to enhance the expression of heterologous proteins by addressing rare codon usage in E. coli. The cells contain the pLysS plasmid, which provides a low level of T7 lysozyme to reduce basal expression of target genes.
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4 protocols using e coli rosetta 2 de3 plyss cells
1
Fluorescent Protein-CBO1751 CBD Fusion
2
Culturing Bacterial Strains for Research
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The bacterial strains used in this study are listed in Supplementary Table S2 . All clostridia were cultured in anaerobic TPGY medium at 30 °C (C. botulinum Group II strains) or 37 °C in an anaerobic workstation with an atmosphere of 85% N2, 10% CO2, and 5% H2 (MK III; Don Whitley Scientific Ltd., West Yorkshire, UK). B. subtilis 1012M15, B. cereus ATCC14579 and E. coli Rosetta 2(DE3) pLysS cells (Merck Millipore, Darmstadt, Germany) were grown in Luria–Bertani (LB) medium at 37 °C. When appropriate, growth media were supplemented with 100 μg/ml ampicillin and 34 μg/ml chloramphenicol.
3
Expression and Purification of Ubiquitin Variants
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Human ubiquitin was expressed using a pET23a-based expression vector in E. coli Rosetta2(DE3) pLysS cells (EMD Millipore) in 2xYT broth by induction with 0.5 mM IPTG and post-induction growth for 4 h at 37°C. Cells were harvested by centrifugation. Purification of the protein was as described elsewhere (24 (link)) with purified protein dialyzed into buffer A (Supplementary Figure S1A). For 15N labeled ubiquitin, the protein was expressed similarly, but in M9 minimal medium supplemented with 15N labeled ammonium chloride (Cambridge Isotope laboratories Inc.) and post-induction growth at 37°C for 14 h.
For PRE experiments, site-directed spin labeling was done at Gly75 of ubiquitin, which was mutated to cysteine. Spin labeling with MTSL and sample preparation was done as described for TDP2 UBA.
Conjugation of monoUb to generate K48- or K63-linked diUbs was done as described elsewhere (24 (link)) with purified conjugated proteins dialyzed into buffer A. Plasmids for all conjugating enzyme expression, pET3d-E2-25K-C170S, pGEX-Ubc13, MBP-His6-TEV-MMS2 and pET21d-Ube1 were obtained from the Addgene plasmid repository. Expression and purification of conjugating enzymes was done as described elsewhere (24 (link)).
For PRE experiments, site-directed spin labeling was done at Gly75 of ubiquitin, which was mutated to cysteine. Spin labeling with MTSL and sample preparation was done as described for TDP2 UBA.
Conjugation of monoUb to generate K48- or K63-linked diUbs was done as described elsewhere (24 (link)) with purified conjugated proteins dialyzed into buffer A. Plasmids for all conjugating enzyme expression, pET3d-E2-25K-C170S, pGEX-Ubc13, MBP-His6-TEV-MMS2 and pET21d-Ube1 were obtained from the Addgene plasmid repository. Expression and purification of conjugating enzymes was done as described elsewhere (24 (link)).
4
Cloning and Purification of CBO1751 Protein
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CBO1751 (GenBank accession number CAL83288) was commercially synthesized and cloned in the NheI/SalI restriction sites of pET21b to generate a C-terminal fusion construct with 6 × His tag (GenScript Biotech, Leiden, Netherlands). The vectors were transformed into E. coli Rosetta 2(DE3) pLysS cells (Merck Millipore). The expression of His-tagged CBO1751 was induced with 1 mM IPTG at 30 °C for 8 h. The expressed protein was purified using metal-chelate affinity chromatography with Ni–IDA resin (Merck Millipore) as previously described51 (link). Eluted proteins were examined by SDS-PAGE prior to dialysis (MWCO 8 kDa, Spectrum Labs, New Brunswick, NJ, USA) against 500 ml of dialysis buffer (DB: 500 mM NaCl, 50% glycerol, 20 mM Tris–HCl, pH 7.9) overnight at 4 °C. Protein concentrations were determined using the Pierce™ BCA Protein Assay Kit (Thermo Fisher Scientific Oy, Vantaa, Finland) with bovine serum albumin (Merck Millipore) as a standard.
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