Cloning of DNA encoding the C-terminal C123 domains of P1 (amino acids 1000–1486) was described previously (Heim et al. 2015 (link)). Recombinant E. coli were grown overnight in 20 mL M9 media and inoculated into 2 L M9 media containing 15N-enriched ammonium chloride and 13C-enriched glucose (Cambridge Isotope Laboratories) as the nitrogen and carbon sources, respectively. Expression was induced at OD600 1.0 with 0.2% L-arabinose. After incubation overnight at 22 °C, cells were harvested by centrifugation at 5400 g for 30 min at 4° C, lysed with an EmulsiFlex-C5 cell disruptor (Avestin), and the cell lysate loaded onto a HisTRAP HP column (GE Healthcare) with 30 mM Tris/100 mM NaCl/10 mM imidazole, pH 7.4, as binding buffer. The rC123 polypeptide was eluted with 30 mM Tris/100 mM NaCl/300 mM imidazole, pH 7.4, and further purified using a HiTrap Q FF column with 15 mM Tris, 10 mM NaCl, pH 8.4, as the binding buffer and 15 mM Tris, 1M NaCl, pH 8.4, as the elution buffer. The eluted product was then polished and exchanged into storage buffer using a HiLoad 16/600 Superdex 200 pg column with 30 mM Tris/100mM NaCl, pH 6.0.
Emulsiflex c5 cell disruptor
Manufactured by Avestin
Sourced in Canada
The EmulsiFlex-C5 is a high-pressure homogenizer designed for the disruption of cells, tissues, and other materials. It uses a high-pressure piston to force the sample through a narrow orifice, creating shear forces that disrupt the target material.
Automatically generated - may contain errors
2 protocols using emulsiflex c5 cell disruptor
1
Cloning and Purification of Isotopically Labeled P1 C123 Domains
2
Expression and Purification of Isotopically Labeled SPOP MATH Domain and Geminin Peptides
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15N-labeled SPOP MATH (amino acids 28–166) harboring a hexahistidine tag cleavable with PreScission protease was expressed in E. coli BL21 (DE3) cells grown in 15NH4Cl-enriched M9 media. The cells were grown at 37 °C to an OD600 of 0.6 and then at 15 °C overnight after addition of isopropyl-β-D-thiogalactoside (IPTG, final concentration 0.5 mM). The harvested cells were lysed using an Avestin Emulsiflex C5 cell disruptor (Avestin Inc., Ottawa, Canada). The protein was purified by nickel affinity chromatography (QIAGEN) and incubated with PreScission protease overnight at 4 °C to cleave the hexahistidine tag. The protein was further purified by size exclusion chromatography on a preparative Superdex 75 16/600 column (GE Healthcare). Using the same protocol, we also purified non-labeled SPOP MATH (D140G mutant34 (link)) for X-ray crystallography. Non-labeled Geminin wild-type SBC (195AEGTVSSSTDAKPCI209) and alanine mutant SBC (195AEGTVAAAADAKPCI209) synthetic peptides were purified by reversed-phase chromatography using a Jupiter 5 μm C18 300 preparative column (Phenomenex). Stocks of the peptides (15 mM) were prepared in the NMR buffer.
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