A synthetic plasmid containing the E. coli codon-optimized gene of R. gnavus E1 encoding RumC1 (pETM-40-rumC1, kanamycin resistant) was obtained from GenScript (Piscataway, NJ), which allows the expression of a MBP (maltose-binding peptide)–tagged peptide. A tobacco etch virus nuclear-inclusion-a endopeptidase (TEV protease) site was inserted in the linker between MBP and RumC1 peptide. pETM40-rumC1 was used to transform competent E. coli BL21 (DE3) cells for expression. The resulting E. coli BL21 (DE3) strain was grown in M9 medium containing kanamycin (50 μg/ml), vitamin B1 (0.5 μg/ml), MgSO4 (1 mM), and glucose (4 mg/ml) at 37°C. At an optical density (OD600) of 0.8, the culture was induced using 1 mM isopropyl-β-d-thiogalactopyranoside (IPTG). The temperature was reduced to 25°C, and the cells were grown for 15 hours under stirring. Cells were then harvested by centrifugation at 4000 rpm for 20 min at 4°C. Cell pellet was suspended in 40 ml of buffer A [50 mM tris (pH 8) and 50 mM NaCl] supplemented with one tablet of a protease inhibitor cocktail (cOmplete, EDTA-free Protease inhibitor cocktail tablets, Roche). Cell pellet was then sonicated, and the lysate was clarified by centrifugation at 40,000 rpm for 40 min at 4°C. The supernatant was collected and passed over Dextrin Sepharose High Performance columns (5 ml; MBPTrap HP, GE Healthcare) coupled to a fast protein liquid chromatography (FPLC) (ÄKTA Purifier 900, ÄKTA FPLC Systems, GE Healthcare). Columns were washed with four column volumes of buffer A. MBP-RumC1 was eluted with buffer B [50 mM tris (pH 8), 50 mM NaCl, and 40 mM maltose]. Fractions containing MBP-RumC1 were pooled and concentrated in a 30,000 molecular weight cutoff (MWCO) filter using Amicon Ultra centrifugal filter devices. The sample was digested by TEV protease for a final TEV protease:MBP-RumC1 ratio of 1:20 (w/w) and incubated for 30 min at room temperature. MBP-tag, TEV protease, and unmodified RumC1 were separated by loading over a HiLoad 16/60 Superdex 75 prep grade column (GE Healthcare) equilibrated in buffer C [50 mM Hepes and 100 mM NaCl (pH 7.5)]. The peptide concentration was estimated by ultraviolet-visible (UV-vis) spectroscopy on a Cary 50 UV-vis spectrophotometer (Varian) by using an extinction coefficient of 8480 M−1 cm−1 at 280 nm.

Note: The content above has been extracted from a research article, so it may not display correctly.

Please log in to submit your questions online.
Your question will be posted on the Bio-101 website. We will send your questions to the authors of this protocol and Bio-protocol community members who are experienced with this method. you will be informed using the email address associated with your Bio-protocol account.

We use cookies on this site to enhance your user experience. By using our website, you are agreeing to allow the storage of cookies on your computer.