Part:BBa_K3515011

Vitamin D receptor binding protein with cysteine modification(s) to bind to a biosensor and FRET to

Vitamin D Receptor Binding Protein (VDR) selectively binds vitamin D in its active site, inducing a conformation change in the N and C termini regions, respectively. This makes it a distinguishable candidate for in vivo or in vitro vitamin D monitoring using fluorescence resonance energy transfer (FRET). Coupling this protein with two fluorophores can permit vitamin D detection. This composite part has coupled VDR with mNeonGreen in the N-terminus and mCherry in the C-terminus regions as FRET pairs. As VDR binds vitamin D these fluorophores in the two terminal regions come together and energy transfer occurs from donor to acceptor, permitting detection as an increase in intensity is detected. Vitamin D detection is vital as vitamin D is used in cells for the regulation of phosphorus and calcium reabsorption, as well as stimulation/activation of proper immune system functioning. Vitamin D is also a crucial biomarker used in clinical medicine for tracking the progression and status of Chronic Kidney Disease (CKD) patients, patients with bone density problems, and patients with cardiovascular complications. As such a biosensor for vitamin D tracking may be of great interest to patients and clinicians. This part includes a mutated VDR to have a cysteine modification that will bind cysteine linker arms and be used for biosensor immobilization allowing the detection of vitamin D. 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.

Vitamin D receptor binding protein fluorescent construct. Protein structures were obtained from the RCSB Protein Data Bank and a construct was made using Chimera software. Torsion angles between fluorescent and binding proteins are adjusted for display purposes. mNeonGreen (green), binding protein (blue), and mCherry (red) are all displayed using a cartoon preset in PyMOL.

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 900
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]