Part:BBa_K3515015

Alpha-Klotho Binding Protein with cysteine modification(s) and FRET to monitor phosphate levels usin

Alpha-Klotho selectively binds FGF23 (peptide hormone) in its active site. A fluorophore may be attached to the N- terminal domain as well to create a FRET signal. This makes it a distinguishable candidate for FGF23 monitoring using fluorescence resonance energy transfer (FRET). Coupling this protein with a supply of FGF23 bound to a second fluorophore can create a competition assay, where FGF23 and FGF23-fluorophore compete with one another for Alpha-Klotho. This composite part has added the Alpha-Klotho protein to mNeonGreen. It would be used in conjunction with a FGF23 peptide hormone bound to mCherry via its terminal amino acid. As these two fluorophores come together FRET can be quantified. If FGF23 from the body competed with FGF23-mCherry it is expected that the FRET signal would decrease. FGF23 detection is vital as FGF23 directly regulates renal function and is also a primary regulator of phosphate and vitamin D suppression. FGF23 is also a crucial biomarker used in clinical medicine for tracking the progression and status of Chronic Kidney Disease (CKD) patients. As such a biosensor for FGF23 tracking may be of great interest to patients and clinicians. This part includes Alpha-Klotho with a cysteine residue that will bind cysteine linker arms and be used for biosensor immobilization allowing the detection of FGF23. Disulfides bonds were not mutated and left in the protein construct. The FRET pair used in this construction were considered especially for physiological detection of phosphate as they have a high intensity and are therefore able to have an expanded dynamic linear range of detection.

Alpha-Klotho (FGF23 receptor) competitive fluorescent assay. Protein structures were obtained from the RCSB Protein Data Bank. All protein residues are shown in a cartoon preset with the mNeonGreen, mCherry, alpha-klotho ternary complex, and FGF23 as green, red, wheat, and grey, respectively, using Chimera software. A. Alpha-Klotho with mNeonGreen and FGF23with mCherry interaction. Fluorescent is expected to occur. B. FGF23 from the interstitial fluid enters the biosensor and competes with the FGF23bound to mCherry. Interaction with alpha-klotho with mNeonGreen. Quantifiable drop in fluorescent intensity expected.

Construct map displaying the entire composite parts coding region. Modifications, linkages, and fluorophore attachment points are described.

A construct map using the pSB1C3 plasmid backbone for illustration purposes.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 2337
  Illegal XhoI site found at 1876
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 871
  Illegal AgeI site found at 1698
  Illegal AgeI site found at 2567
  
• 1000
  COMPATIBLE WITH RFC[1000]