pET28a-6xHis-TrxF
(Plasmid #207454)

• Purpose: E. coli expression of Mesembryanthemum crystallinum Thioredoxin F with thrombin-cleavable N-terminal 6xHis tag
• Depositing Labs: Joseph Jez, Sarah Stainbrook
• Publication: Stainbrook et al bioRxiv 2022.08.24.505184

Backbone

• Vector backbone: pET28a
• Backbone size w/o insert (bp): 5293
• Total vector size (bp): 5827
• Modifications to backbone: None
• Vector type: Bacterial Expression

Growth in Bacteria

• Bacterial Resistance(s): Kanamycin, 50 μg/mL
• Growth Temperature: 37°C
• Growth Strain(s): DH5alpha
• Copy number: Unknown

Gene/Insert

• Gene/Insert name: Thioredoxin F
• Alt name: TrxF
• Species: Synthetic; Mesembryanthemum crystallinum
• Insert Size (bp): 534
• Mutation: Codon optimized for E coli expression
• Promoter: T7
• Tag / Fusion Protein:
  • 6xHis (N terminal on insert)

Cloning Information

• Cloning method: Restriction Enzyme
• 5′ cloning site: NheI (not destroyed)
• 3′ cloning site: xhoI (not destroyed)
• 5′ sequencing primer: T7 promoter
• 3′ sequencing primer: T7 terminator

Resource Information

• Supplemental Documents:
  • pet28a-6xhis-trxf.gb

Terms and Licenses

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

Depositor Comments

Expression protocol: Transformed BL21-DE3 was grown to OD600 0.6-0.8, induced with 1mM IPTG and incubated at 12oC overnight. TrxF was purified by Ni-NTA affinity chromatography in buffer containing 50mM HEPES pH 8.0, 0.5M NaCl, 5% glycerol, 10mM β-mercaptoethanol and 5-300mM imidazole, then dialyzed into the same buffer without imidazole and stored in aliquots at -80oC. TrxF activity was verified using the insulin reduction assay (Holmgren, 1979).

Please visit https://www.biorxiv.org/content/10.1101/2022.08.24.505184v1for bioRxiv preprint.