Publication protocol
"For the expression of each clone, a single positive colony was selected from a freshly transformed plate and grown in 100-mL LB broth, with kanamycin (50 µg/mL, for a protein gene on pRSF1b vector), ampicillin (100 µg/mL, for a protein gene on pBAD/HisB vector), or both (cotransformation) used as selection markers. Following overnight incubation at 37 °C, 12.5 mL of the starter culture was used to inoculate a 500-mL LB broth and allowed to grow until an absorbance (OD600) of 0.4–0.5 was reached. Protein expression was then induced with 0.4 mM IPTG (pRSF vector), 0.1% L-arabinose (pBAD vector), or both (cotransformation). We tested the role of a tightly controlled relative expression of the encapsulated POI by evaluating the effect of L-arabinose (0.01, 0.1, and 1%) on the functional expression of GFPuv. After 4 h of incubation at 37 °C, cells were pelleted by centrifugation at 13,750 ×g for 10 min. The cell pellet was resuspended in a lysis buffer (25 mM Tris-HCl, 150 mM NaCl, pH 7.5), sonicated, and centrifuged to separate cell debris. The supernatant obtained was purified using a two-step chromatography procedure. pRSF clones were subjected to hydrophobic interaction chromatography (HIC) using HiPrepTM Phenyl FF (low sub) 16/10 (GE healthcare), followed by SEC using a Superdex S-200 10/300 GL column (GE healthcare). pBAD clones were subjected to Ni-NTA (Expedeon) column chromatography, followed by SEC. Fusion proteins were purified using Ni-NTA and SEC. The purity of the SEC fraction was analyzed using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Buffers used were as follows: HIC: buffer A, 25 mM Tris-HCl, 150 mM NaCl, and 1 M (NH4)2SO4, pH 7.5; buffer B, 25 mM Tris-HCl, 150 mM NaCl, pH 7.5; Ni-NTA chromatography: buffer A, 25 mM Tris-HCl, 150 mM NaCl, pH 7.5; buffer B, 25 mM Tris-HCl, 150 mM NaCl, and 500 mM imidazole, pH 7.5; and SEC: 25 mM Tris-HCl, pH 8. All buffers used for the lysis, purification, and assays of HRPc clones were supplemented with 5 mM CaCl2 and 2.5 µM hemin (a stock solution of 40 mM was prepared by dissolving 25 mg of hemin in 1 mL of 1.4 N ammonium hydroxide).
"
Full paper
Login or
join for free to view the full paper.
Manufacturer protocol
Download the product protocol from Chester L. Drum, Cardiovascular Research Institute, Department o for pRSF1b below.
We haven't found the manufacturer protocol for this product yet.
Videos
Check out videos that might be relevant for performing Protein Expression Prokaryotic cells - E. coli AfFtn using pRSF1b from Chester L. Drum, Cardiovascular Research Institute, Department o. Please note that these videos are representative and steps or experiment specific processes must be kept in mind to expect desired results.
We haven't found any additional videos for this experiment / product combination yet.