pET21b-LOV-ipaA L514K L531E
(Plasmid #40253)

• Depositing Lab: Brian Kuhlman
• Publication: Lungu et al Chem Biol. 2012 Apr 20;19(4):507-17.

Backbone

• Vector backbone: pET-21b(+)
• Backbone manufacturer: EMD Biosciences
• Backbone size w/o insert (bp): 5442
• Total vector size (bp): 5939
• 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: LOV-ipaA L514K L531E
• Alt name: Light-sensitive LOV2 domain of Avena Sativa phototropin 1(AsLov2)/Shigella invasin protein ipaA vinculin binding peptide chimera
• Alt name: AsLOV2 (aa 404-546)
• Alt name: ipaA (aa 610-631)
• Species: Synthetic; Avena sativa/Shigella flexneri
• Insert Size (bp): 497
• Mutation: L514K; L531E; Jα helix region hybridized (AsLOV2 aa 537-546/ipaA aa 610-619), residue 540 changed to isoleucine
• GenBank ID: AAC05083.1 AAP78988.1
• Promoter: T7
• Tag / Fusion Protein:
  • 6xHis (N terminal on insert)

Cloning Information

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

Terms and Licenses

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

Depositor Comments

This vector encodes a photo-activable caged form of the vinculin binding ipaA peptide. It contains a set of mutations, L514K L531E, that increases the dynamic range of the switch. These mutations replace two hydrophobic residues, one in the Jα helix, and the other on the β sheet contacting the Jα helix, with a salt bridge. This design stabilizes interactions between the β-sheet and Ja helix and leads to a more tightly bound helix in the dark state. This set of mutations makes LOV-ipaA a photoswitch with a 49-fold difference between the lit- and dark-state effector binding affinities.

Regarding the hybridized region, the first ten residues of the ipaA VBS1 helical peptide were identified as a close match to the last ten residues of the AsLOV2 Jα helix; five of the ten positions are identical, and the hydrophobic residues on the Jα helix that make critical contacts with the AsLOV2 domain β-sheet at residues 539, 542, and 543 are conserved in the alignment with ipaA. Additionally, residues in the ipaA sequence that make extensive contacts with vinculin are conserved in the alignment (Ile 612, Ala 615, Ala 616, and Val 619 in ipaA). Side-chain optimization simulations were used to thread the first ten residues of ipaA onto the last ten residues of the Jα helix. The 540 position on the Jα helix was converted to isoleucine.

The LOV-ipaA gene was synthesized with a six histidine N-terminal tag (Genscript, Piscataway, NJ, USA) and cloned into the pET21b vector. All mutations were performed using site-directed mutagenesis.

Protein coding sequence of LOV-ipaA L514K L531E:
MHHHHHHGSLATTLERIEKNFVITDPRLPDNPIIFASDSFLQLTEYSREEILGRNCRFLQGPETDRATVRKIRDAIDNQTEVTVQLINYTKSGKKFWNLFHLQPMRDQKGDVQYFIGVQKDGTEHVRDAAEREGVMEIKKTANNIIKAAKDVTTSLSKVLKNIN