pET21b-LOV-ipaA L514K L531E
|Item||Catalog #||Description||Quantity||Price (USD)|
|Plasmid||40253||Standard format: Plasmid sent in bacteria as agar stab||1||$65|
This material is available to academics and nonprofits only.
Backbone manufacturerEMD Biosciences
- Backbone size w/o insert (bp) 5442
- Total vector size (bp) 5939
Vector typeBacterial Expression
Growth in Bacteria
Copy numberHigh Copy
Gene/Insert nameLOV-ipaA L514K L531E
Alt nameLight-sensitive LOV2 domain of Avena Sativa phototropin 1(AsLov2)/Shigella invasin protein ipaA vinculin binding peptide chimera
Alt nameAsLOV2 (aa 404-546)
Alt nameipaA (aa 610-631)
SpeciesSynthetic; Avena sativa/Shigella flexneri
Insert Size (bp)497
MutationL514K; L531E; Jα helix region hybridized (AsLOV2 aa 537-546/ipaA aa 610-619), residue 540 changed to isoleucine
GenBank IDAAC05083.1 AAP78988.1
- Promoter T7
/ Fusion Protein
- 6xHis (N terminal on insert)
- 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 (Common Sequencing Primers)
Terms and Licenses
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:
These plasmids were created by your colleagues. Please acknowledge the Principal Investigator, cite the article in which the plasmids were described, and include Addgene in the Materials and Methods of your future publications.
For your Materials & Methods section:pET21b-LOV-ipaA L514K L531E was a gift from Brian Kuhlman (Addgene plasmid # 40253 ; http://n2t.net/addgene:40253 ; RRID:Addgene_40253)
For your References section:Designing photoswitchable peptides using the AsLOV2 domain. Lungu OI, Hallett RA, Choi EJ, Aiken MJ, Hahn KM, Kuhlman B. Chem Biol. 2012 Apr 20;19(4):507-17. 10.1016/j.chembiol.2012.02.006 PubMed 22520757