pET24a_UDP_Xase
(Plasmid #73158)

• Purpose: Expresses in E. coli, S. pneumoniae TIGR4 UDP-Xase SP_1669
• Depositing Lab: Sandra Gabelli
• Publication: Duong-Ly et al PLoS One. 2013 May 15;8(5):e64241. doi: 10.1371/journal.pone.0064241. Print 2013.

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: For protein production, use BL21 DE3 cells. Cells were grown at 37°C in LB media. When an OD600 of 0.9 was reached, 1 mM IPTG was added to induce expression. After overnight growth at 22°C, the cells were centrifuged, washed in 50 mM Tris pH 7.5, 1 mM EDTA, and 0.1 mM DTT (TED buffer), reharvested, and stored at −80°C.
• Copy number: Low Copy

Gene/Insert

• Gene/Insert name: pET24a
• Alt name: A0A0H2URA0
• Alt name: NC_003028.3
• Alt name: Sp_1699
• Species: Streptococcus pneumoniae serotype 4 (strain ATCC BAA-334 / TIGR4
• GenBank ID: NC_003028.3

Terms and Licenses

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

Depositor Comments

The over-expressed protein was released upon thawing of the
cells. The cell extract was adjusted to 33% ammonium sulfate
saturation and allowed to precipitate for 30 minutes at 4uC.
Precipitated protein was harvested by centrifugation at 4uC for 30minutes at 172006g on a GSA rotor (Sorvall). TED buffer was
used to resuspend the precipitated protein. The remaining soluble
protein was brought up to 50% ammonium sulfate saturation and
the above procedure was repeated to harvest and resuspend
precipitated protein. The supernatant after this step was brought
up to 30% glycerol (v/v) and loaded onto a Sephadex, G-100
(2.5650 cm) gel filtration column equilibrated with TED buffer
containing 100 mM NaCl.

THe structure has been determined by X-ray crystallography and deposited in the PDB 4HFQ