pTEV5_ RpMatB_4
(Plasmid #78208)

• Purpose: Expression of double-cysteine variant of Malonyl-coenzyme A synthetase, RpMatB, as scaffold for ATP biosensor - weak binding
• Depositing Lab: Martin Webb
• Publication: Vancraenenbroeck et al PLoS One. 2017 Jun 21;12(6):e0179547. doi: 10.1371/journal.pone.0179547. eCollection 2017.

Backbone

• Vector backbone: pTEV5
• 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 OverExpress C41(DE3) for protein expression
• Copy number: High Copy

Gene/Insert

• Gene/Insert name: matB (His6 C106A R286C T303A Q457C K488A)
• Species: Rhodopseudomonas palustris
• Mutation: C106A R286C T303A Q457C K488A
• Promoter: T7
• Tag / Fusion Protein:
  • His6 (N terminal on backbone)

Cloning Information

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

Resource Information

• Supplemental Documents:
  • pTEV5_ RpMatB_4 information
  • pTEV5_ RpMatB_4 protocol

Terms and Licenses

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

Depositor Comments

The plasmid is used to express Malonyl-coenzyme A synthetase from Rhodopseudomonas palustris (RpMatB), containing a His-tag and the mutations C106A, R286C, T303A, Q457C and K488A: (His6/C106A/R286C/Q457C/K488A/T303A)RpMatB.

The protein is modified for use as the scaffold of an ATP biosensor, Rho-RpMatB(T303A). The two cysteine mutations are for label attachment (R286C and Q457C); there is a mutation to prevent enzyme activity (K488A), a mutation to change the wild-type cysteine (C106A), a mutation that weakens ATP binding (T303A) and an N-terminal His-tag to aid purification.

When labelled with two 5-iodoacetamidotetramethylrhodamine (5-IATR) fluorophores, this adduct can be used as an ATP biosensor has a fluorescence intensity change of ~3-fold. With a Kd of ~40 μM, this adduct variant can be used to measure ATP formation in the range of up to ~100 μM, in the presence of millimolar ADP.