pET24_BD3179
(Plasmid #73142)

• Purpose: Expresses in E.coli B. bacteriovorus Bd3179
• Depositing Lab: Sandra Gabelli
• Publication: de la Peña et al PLoS One. 2015 Nov 2;10(11):e0141716. doi: 10.1371/journal.pone.0141716. eCollection 2015.

Backbone

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

Growth in Bacteria

• Bacterial Resistance(s): Kanamycin, 50 μg/mL
• Growth Temperature: 37°C
• Growth Strain(s): DH5alpha
• Growth instructions: BL21 (DE3) cells were transformed with a pET24a (Novagen, Madison WI) plasmid containing the Bdellovibrio bacteriovorus Bd3179 gene. Cells were grown at 37°C from a single colony inoculum in LB media supplemented with Kanamycin. Bd-NDPSase expression was induced at an OD600 of 0.7 with 100 μM IPTG. Cells were grown for three to five hours, centrifuged, pelleted, and frozen at -80°C.
• Copy number: Low Copy

Gene/Insert

• Gene/Insert name: Bd3179
• Alt name: Q6MIH8 UNIPROT, NUDF
• Alt name: CAE80935; CAE80935; Bd3179.
• Species: Bdellovibrio bacteriovorus HD 100
• GenBank ID: NC_005363

Resource Information

• Supplemental Documents:
  • Structural and Enzymatic Characterization of a Nucleoside Diphosphate Sugar Hydrolase from Bdellovibrio bacteriovorus.

Terms and Licenses

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

Depositor Comments

The cell pellet was thawed, resuspended in 80 ml TE buffer (50 mM Tris-HCl pH 7.5, 1 mM EDTA), and lysed with a microfluidizer 2x at 75 psi. The cell lysate was clarified by centrifugation for 30 minutes at 11,500 rpm using TE buffer at 0, 30, and 60% (w/v) ammonium sulfate concentrations. The pellet from the last clarification step was resuspended in TE buffer at a 60% (w/v) ammonium sulfate concentration, filtered with a Corning PES 0.20 μm filter, and loaded on a hydrophobic interaction column (Phenyl FF HiPrep 16/10, GE Healthcare) equilibrated in resuspension buffer. Bound protein was eluted with a TE buffer gradient. Fractions containing protein were dialyzed at 4°C 2x against 2 L of 50 mM Tris-HCl pH 8.6, 1 mM EDTA buffer overnight in 3500 Da dialysis tubing (SnakeSkin, Thermo Scientific), and loaded onto an anion exchange column (Resource Q, GE Healthcare). The protein was loaded with 50 mM Tris-HCL pH 8.6, 1 mM EDTA (Buffer A) and eluted with a 50 mM Tris-HCl pH 8.5, 1 mM EDTA and 1 M NaCl (Buffer B) gradient.

The structure was determined by xray crystallography and deposited in the PDB 5C7Q, 5C7T, 5C8L