The WT A3A was purified as described in Sharma et al. (2015) (link). Briefly, Rosetta 2(DE3)pLysS E. coli (EMD Millipore, Burlington, MA, USA) transformed with a bacterial expression construct for C-terminal His6-tagged WT A3A was grown in Luria broth at 37 °C. The cells were induced for expression of the recombinant protein with 0.3 mM isopropyl β-D-1-thiogalactopyranoside and cultured overnight at 18 °C. A3A protein was purified from the lysates by affinity chromatography using the Ni-NTA His bind Resin (EMD Millipore). The concentrated protein was stored in 25 mM Tris (pH 8.0) with 50 mM NaCl, 1 mM DTT, 5% v/v glycerol and 0.02% w/v sodium azide at −80 °C.
Rosetta 2 de3 plyss e coli
Manufactured by Merck Group
Sourced in United States
Rosetta 2(DE3)pLysS E. coli is an E. coli expression strain designed for the expression of proteins that contain codons rarely used in E. coli. The strain carries the pLysS plasmid, which provides low-level expression of T7 lysozyme to reduce basal expression of target genes.
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3 protocols using rosetta 2 de3 plyss e coli
1
Purification of Recombinant WT A3A Protein
2
Recombinant Protein Expression in E. coli
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This construct was transformed into Rosetta 2 (DE3) pLysS E. coli (Novagen; Merck & Co., Inc.) using a chemical method. Plasmid DNA was added to 100 µl competent cells on ice. The whole mixture was incubated on ice for 30 min. The bacteria were shocked at 42°C and cooled on ice. LB medium was added and culture was grown at 37°C for 45 min. The transformation mix was transferred onto LB agar supplemented with ampicillin (100 µg/ml) and chloramphenicol (34 µg/ml). E. coli was routinely grown overnight at 37°C, with standard antibiotic plate selection performed according to the manufacturer's instructions. Transformed E. coli were grown overnight in LB media supplemented with ampicillin (100 µg/ml) and chloramphenicol (34 µg/ml). Cultures were then diluted in a 1:100 ratio in the same media and cultured at 37°C until they reached an optical density reading of between 0.6 and 0.8 at a wavelength of 600 nm. Then, protein expression was induced with 1 mmol/l IPTG and cultures were grown for 16 h at 37°C.
3
Expression and Purification of APOBEC3A
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Rosetta 2(DE3)pLysS E. coli (EMD Millipore) transformed with a bacterial expression construct for C-His6-tagged APOBEC3A and grown in Luria broth at 37 °C were induced for expression of the recombinant protein with 0.3 mM isopropyl β-D -1-thiogalactopyranoside and cultured overnight at 18 °C. Harvested cells were lysed with a French pressure cell (American Instrument Corporation, Hartland, WI) and Ni-NTA His.Bind Resin (EMD Millipore) was used as per manufacturer's instructions to purify APOBEC3A protein from the lysates by affinity chromatography. Isolated protein was concentrated using an Amicon Ultra-4 Centrifugal Filter Unit with Ultracel-3 membrane (EMD Millipore; nominal molecular weight limit of 3 kDa). The concentrated protein was stored in 25 mM Tris (pH 8.0) with 50 mM NaCl, 1 mM DTT, 5% v/v glycerol and 0.02% w/v sodium azide. Staining with Coomassie blue of protein preparation electrophoresed on a denaturing polyacrylamide gel indicated that it had APOBEC3A at >90% purity.
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