BPV00914 pET21GG2 H6-TF-BECN (1-450) [VA-MA-LA]
(Plasmid #136688)

• Purpose: Expresses the full-length Beclin-1 protein (a.a. 1-450) with a N-terminal hexa-Histidine and solubility tag (i.e., Trigger Factor). Three mutations are added to the CC and ECD/BARA interface.
• Depositing Lab: Colin Garvie
• Publication: Ranaghan et al Biochemistry. 2017 Dec 26;56(51):6639-6651. doi: 10.1021/acs.biochem.7b00936. Epub 2017 Dec 14.

Backbone

• Vector backbone: pET21b
• Total vector size (bp): 8208
• Vector type: Bacterial Expression

Growth in Bacteria

• Bacterial Resistance(s): Ampicillin, 100 μg/mL
• Growth Temperature: 37°C
• Growth Strain(s): DH5alpha
• Growth instructions: Large-scale expression of BECN1 fusion proteins was performed by growing cultures in TB at 37 °C with orbital shaking at 180 rpm to mid-log phase (A600 ∼ 0.6−0.8), cooling cultures to 18 °C for 30 min before induction with 0.5 mM isopropyl β-D-1-thiogalactopyranoside, and growing the induced cultures for 18 h at 18 °C. Cell pellets were harvested via centrifugation and stored at −50 °C.
• Copy number: Unknown

Gene/Insert

• Gene/Insert name: Beclin-1
• Alt name: BECN
• Species: H. sapiens (human)
• Insert Size (bp): 2830
• Mutation: DNA optimized for E. coli expression w/ three mutations [V250A-M254A-L261A]
• GenBank ID: AF139131 AF139131
• Entrez Gene: BECN1 (a.k.a. ATG6, VPS30, beclin1)
• Promoter: T7
• Tag / Fusion Protein:
  • Trigger Factor (N terminal on insert)

Cloning Information

• Cloning method: Restriction Enzyme
• 5′ cloning site: BsaI (destroyed during cloning)
• 3′ cloning site: BsaI (destroyed during cloning)
• 5′ sequencing primer: T7 promoter
• 3′ sequencing primer: T7 terminator

Resource Information

• A portion of this plasmid was derived from a plasmid made by: Synthesized via GeneArt

Terms and Licenses

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

Depositor Comments

Large-scale expression of the BECN1 fusion protein was performed by growing cultures in TB at 37 °C with orbital shaking at 180 rpm to mid-log phase (A600 ∼ 0.6−0.8), cooling cultures to 18 °C for 30 min before induction with 0.5 mM isopropyl β-D-1-thiogalactopyranoside, and growing the induced cultures for 18 h at 18 °C. Cell pellets were harvested via centrifugation and stored at −50 °C.

See Ranaghan et al. (2017) [DOI: 10.1021/acs.biochem.7b00936] for purification methods.