pET24a_UDP_Xase_E162
(Plasmid #73160)

• Purpose: Expresses in E. coli, S. pneumoniae TIGR4 UDP-Xase SP_1669 mutant E162A
• 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 GC5 cells.
• 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
• Mutation: change glutamate 162 to alanine
• GenBank ID: NC_003028.3

Cloning Information

• Cloning method: Restriction Enzyme
• 5′ cloning site: BAMH1 (not destroyed)
• 3′ cloning site: NdeI (not destroyed)
• 5′ sequencing primer: 5 9 -TGGACAATTCCAA- GAAAATCAAGCAATTGCTGACCTTCAATTTTTTG-3
• 3′ sequencing primer: 5 9 -CAAAAAATTGAAGGTCAGCAATTGCTT- GATTTTCTTGGAATTGTCCA-3 9 ,

Terms and Licenses

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

Depositor Comments

Cells expressing proteins containing mutations were lysed by
microfludization. The lysate was brought up to 30% ammonium
sulfate saturation, proteins were precipitated, and then resus-
pended in TED buffer. The remaining lysate was brought to 60%
ammonium sulfate saturation and proteins were precipitated and
resuspended, brought to 90% ammonium sulfate saturation and
precipitated and resuspended again. The proteins of interest were
present in the 60% and 90% ammonium sulfate precipitates.
These protein fractions were further purified by hydrophobic
interaction chromatography using a Phenyl FF 16/10 High Sub
column (GE Healthcare) followed by anion exchange chromatog-
raphy on a Source Q column (GE Healthcare) and size exclusion
chromatography on a Sephacryl S200 16/60 column (GE
Healthcare).