pGEX-optimized E6AP F727D
(Plasmid #165101)

• Purpose: ecoli optimized expression of a GST fusion of Human E6AP F727D
• Depositing Lab: Arthur Haas
• Publication: Ronchi et al J Biol Chem. 2014 Jan 10;289(2):1033-48. doi: 10.1074/jbc.M113.517805. Epub 2013 Nov 22.

Backbone

• Vector backbone: pGEX4T1
• Backbone manufacturer: GE Healthcare
• Backbone size w/o insert (bp): 4900
• Vector type: Bacterial Expression

Growth in Bacteria

• Bacterial Resistance(s): Ampicillin, 100 μg/mL
• Growth Temperature: 37°C
• Growth Strain(s): DH5alpha
• Copy number: High Copy

Gene/Insert

• Gene/Insert name: E6AP F727D
• Alt name: UBE3A
• Species: H. sapiens (human)
• Insert Size (bp): 2620
• Mutation: codon optimized for E. coli expression
• Entrez Gene: UBE3A (a.k.a. ANCR, AS, E6-AP, EPVE6AP, HPVE6A, PIX1)
• Tag / Fusion Protein:
  • GST from plasmid. (N terminal on insert)

Cloning Information

• Cloning method: Ligation Independent Cloning

Terms and Licenses

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

Depositor Comments

Amino acid numbering is based on Isoform 1 though this clone is isoform 3. The sequence is 5'-SPLKYLFRPEE-3' where F has been mutated to D. Best yields for protein purification were found in BL21 (DE) cells using an overnight induction at 16 degrees C.

5' Cloning Site: EcoRI (not destroyed), 3' Cloning Site: XhoI (not destroyed).