pSVA937

Protocol tips

Upstream tips	Protocol tips	Downstream tips
	E. coli Rosetta DE3 pLysS chemically competent cells (Novagen, Merck KGaA, Darmstadt, Germany) were transformed with the codon-optimized MalR expression plasmid pSVA937. Four 1-liter volumes of LB medium containing 0.5% glucose, 50 μg/ml ampicillin, and 30 μg/ml chloramphenicol were each inoculated with 30 ml preculture and induced with 0.5 mM isopropyl-β-d-thiogalactopyranoside (IPTG) when cells reached an OD600 of 1.

Protocol tips

E. coli Rosetta DE3 pLysS chemically competent cells (Novagen, Merck KGaA, Darmstadt, Germany) were transformed with the codon-optimized MalR expression plasmid pSVA937. Four 1-liter volumes of LB medium containing 0.5% glucose, 50 μg/ml ampicillin, and 30 μg/ml chloramphenicol were each inoculated with 30 ml preculture and induced with 0.5 mM isopropyl-β-d-thiogalactopyranoside (IPTG) when cells reached an OD600 of 1.

Publication protocol

E. coli Rosetta DE3 pLysS chemically competent cells (Novagen, Merck KGaA, Darmstadt, Germany) were transformed with the codon-optimized MalR expression plasmid pSVA937. Four 1-liter volumes of LB medium containing 0.5% glucose, 50 μg/ml ampicillin, and 30 μg/ml chloramphenicol were each inoculated with 30 ml preculture and induced with 0.5 mM isopropyl-β-d-thiogalactopyranoside (IPTG) when cells reached an OD600 of 1. Cells were harvested after 2.5 h of induction and were disrupted by sonication in buffer C (50 mM Tris-HCl [pH 8.0], 200 mM NaCl, 1 mM dithiothreitol [DTT], 2 mM EDTA). Cell debris was removed by centrifugation at 25,000 × g, and soluble protein was precipitated with 80% saturated ammonium sulfate. The precipitated protein was resuspended in buffer C supplied with 5 mM imidazole. Precipitates were removed by centrifugation at 236,400 × g. The supernatant was loaded onto 1 ml cOmplete His-tag purification resin (Roche Diagnostics GmbH, Mannheim, Germany), preequilibrated with buffer C containing 5 mM imidazole. The column was washed with buffer C containing 50 mM imidazole and was eluted with buffer C containing 300 mM imidazole. The eluted fraction was pooled by buffer exchange with an Amicon Ultra-4 10K device (Millipore, Merck KGaA, Darmstadt, Germany) using buffer D (50 mM Tris-HCl [pH 8.0], 120 mM NaCl, 10% glycerol, 1 mM DTT, 2 mM EDTA) to remove imidazole.

Full paper   Login or join for free to view the full paper.

Reviews

Discussion

Papers

Check out relevant papers found by Labettor's AI that are relevant for performing Protein Expression Prokaryotic cells - E. coli MalR using pSVA937 from Sonja-Verena Albers, Molecular Biology of Archaea, Max Planck In.

Manufacturer protocol

Download the product protocol from Sonja-Verena Albers, Molecular Biology of Archaea, Max Planck In for pSVA937 below.

Videos

Check out videos that might be relevant for performing Protein Expression Prokaryotic cells - E. coli MalR using pSVA937 from Sonja-Verena Albers, Molecular Biology of Archaea, Max Planck In. Please note that these videos are representative and steps or experiment specific processes must be kept in mind to expect desired results.