pET24a_CDP-CHase
(Plasmid #73161)

• Purpose: Expresses in E. coli, Bacillus cereus NP_831800 CDP-CHase
• Depositing Labs: Maurice Bessman, Sandra Gabelli
• Publication: Duong-Ly et al J Bacteriol. 2011 Jul;193(13):3175-85. doi: 10.1128/JB.00089-11. Epub 2011 Apr 29.

Backbone

• Vector backbone: pET24
• Backbone manufacturer: Novagen
• Vector type: Bacterial Expression

Growth in Bacteria

• Bacterial Resistance(s): Kanamycin, 50 μg/mL
• Growth Temperature: Room Temperature
• Growth Strain(s): DH5alpha
• Growth instructions: For protein production, use BL21 DE3 cells. Cells were grown at 37°C in LBmedium supplemented with 30g/ml kanamycin to an optical density of 0.8 at 600 nm, induced with 0.1 mM isopropyl-D-1-thiogalactopyranoside (IPTG),and grown at 15°C overnight.
• Copy number: Low Copy

Gene/Insert

• Gene/Insert name: pET24a
• Alt name: Q81EE8
• Alt name: NP831800
• Species: Bacillus cereus ATCC 14579
• GenBank ID: NC_004722.1

Resource Information

• Article Citing this Plasmid:
  • 1 Reference

Terms and Licenses

• Academic/Nonprofit Terms:
  • UBMTA
• Industry Terms:
  • Not Available to Industry

Depositor Comments

Cells were harvested via centrifugation, resus-pended in 50 mM Tris-HCl, pH 7.5, 1 mM EDTA, and 0.1 mMdithiothreitol(DTT) (TED), and frozen at -80°C. Following thawing, cells were lysed bymicrofluidization and cell debris was removed by centrifugation. The lysate wasfractionated by first bringing the solution to 30% saturation, discarding thepellet, and then subsequently increasing the saturation to 60% and 90% with(NH4)2SO4and dissolving the precipitates in TED. The protein was further
purified using hydrophobic interaction chromatography over a HiPrep 16/10phenyl FF (high sub) column (GE Healthcare), dialyzed against TED, andsubjected to anion exchange chromatography over a Source Q column (GEHealthcare). The eluted protein was passed over a HiPrep 26/60 Sephacryl S100HR column (GE Healthcare) for size exclusion.

The structure was determined by X-ray Crystallography and deposited in the PDB as 3Q1P 3Q4I