pET22b_PstS_2
(Plasmid #78199)

• Purpose: Expression of double-cysteine variant of mature phosphate binding protein, PstS, as scaffold for phosphate biosensor
• Depositing Lab: Martin Webb
• Publication: Solscheid et al Biochemistry. 2015 Aug 18;54(32):5054-62. doi: 10.1021/acs.biochem.5b00449. Epub 2015 Aug 4.

Backbone

• Vector backbone: pET22b
• Vector type: Bacterial Expression

Growth in Bacteria

• Bacterial Resistance(s): Ampicillin, 100 μg/mL
• Growth Temperature: 37°C
• Growth Strain(s): DH5alpha
• Growth instructions: use BL21 (DE3) for protein expression
• Copy number: High Copy

Gene/Insert

• Gene/Insert name: pstS (A17C,A197C)
• Species: E. coli
• Mutation: A17C; A197C (please see depositor comment below)
• Promoter: T7

Cloning Information

• Cloning method: Restriction Enzyme
• 5′ cloning site: NdeI (unknown if destroyed)
• 3′ cloning site: XhoI (unknown if destroyed)
• 5′ sequencing primer: T7
• 3′ sequencing primer: T7 terminal

Resource Information

• Supplemental Documents:
  • pET22b_PstS_2 protocol
  • pET22b_PstS_2 information

Terms and Licenses

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

Depositor Comments

Please note- The numbering system used was for the mature natural wild-type PstS protein. The plasmid produces an extra N-terminal methionine so all mutations will be off by 1nt (e.g., A197C =A198C)

The expressed protein is a double-cysteine variant of E. coli phosphate binding protein. The gene encodes the mature protein without the N-terminal signal peptide, but with an N-terminal methionine, for subsequent labeling at the cysteines with two 6-iodoacetamidotetramethylrhodamines per molecule. This adduct is then used as a fluorescent phosphate biosensor, having 10-15-fold fluorescence increase on binding inorganic phosphate with very high affinity.