Part:BBa_K3515013

Parathyroid Hormone Receptor with cysteine modification(s) and FRET to monitor PTH levels using a b

The parathyroid hormone receptor (PTHR) selectively binds parathyroid hormone (PTH) in its N- terminal extracellular domain. A fluorophore may be attached to the N- terminal domain as well to create a FRET signal. This makes it a distinguishable candidate for PTH monitoring using fluorescence resonance energy transfer (FRET). Coupling this protein with a supply of PTH bound to a second fluorophore can create a competition assay, where PTH and PTH-fluorophore compete with one another for PTHR. This composite part has added the N- terminal extracellular domain of PTHR to mNeonGreen. It would be used in conjungtion with a PTH peptide fragment bound to mCherry via its lysine amino acid side chain. As these two fluorophores come together FRET can be quantified. If PTH from the body competed with PTH-mCherry it is expected that the FRET signal would decrease. PTH detection is vital as PTH directly regulates renal function and is also a primary regulator of calcium levels. PTH 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 PTH tracking may be of great interest to patients and clinicians. This part includes the N- terminal extracellular domain of PTHR with a cysteine residue that will bind cysteine linker arms and be used for biosensor immobilization allowing the detection of PTH. 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.

Parathyroid hormone 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, parathyroid hormone receptor, and parathyroid hormone (1-34) as green, red, blue, and grey, respectively, using Chimera software. A. Parathyroid receptor hormone with mNeonGreen and parathyroid hormone with mCherry interaction. Fluorescent is expected to occur. B. Parathyroid hormone from the interstitial fluid enters the biosensor and competes with the parathyroid hormone bound to mCherry. Interaction with parathyroid hormone receptor 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 821
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 737
  
• 1000
  COMPATIBLE WITH RFC[1000]