FN(9-10, RGE)-I27-CC
(Plasmid #85396)

• Purpose: MTFM mechanosensor for integrin. Fibronectin domains 9&10 with RGE mutation fused to titan I27 domain with two cysteines for immobilization, site for p-azidophenylalanine incorporation & Cy3 labeling
• Depositing Lab: Khalid Salaita
• Publication: Galior et al Nano Lett. 2016 Jan 13;16(1):341-8. doi: 10.1021/acs.nanolett.5b03888. Epub 2015 Dec 7.

Backbone

• Vector backbone: pET-22b
• Backbone manufacturer: Novagen
• Backbone size w/o insert (bp): 5493
• Vector type: Bacterial Expression

Growth in Bacteria

• Bacterial Resistance(s): Ampicillin, 100 μg/mL
• Growth Temperature: 30°C
• Growth Strain(s): DH5alpha
• Growth instructions: Use BL21(DE3) strain for expression
• Copy number: Unknown

Gene/Insert

• Gene/Insert name: Fibronectin (FN) domains 9&10 containing RGE mutation fused to titin immunoglobulin domain (I27), containing a TAG (amber) codon
• Alt name: KS32-ECC
• Mutation: GRGDS motif mutated to GRGES
• Promoter: T7
• Tag / Fusion Protein:
  • 6x His (C terminal on backbone)

Cloning Information

• Cloning method: Unknown
• 5′ sequencing primer: T7
• 3′ sequencing primer: T7-term

Resource Information

• Supplemental Documents:
  • 85396_genbank

Terms and Licenses

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

Depositor Comments

Fibronectin (FN) domain 9 and 10 containing PHSRN and mutant GREDS motif, which prevents unfolding of I27.

A TAG (amber) codon was added between the FN 9-10 domains and I27 for labeling with p-azidophenylalanine, an unnatural amino acid. The FN(9-10)-I27-CC fusion protein can be labeled with the Cy3 dye at this site using Cu-free click cyanoaddition coupling.

I27 domain is the 27th immunoglobulin domain of cardiac titin. Two cysteine residues were added at the C-terminus of I27 to allow immobilization onto a gold nanoparticle.

Please see the associated publication for material and methods information.