Ribose is essential for synthesizing nucleotides, the molecules that build DNA, but for how important this sugar is, there is much to still be learned about ribose within mammalian cells. For instance, uncertainty still remains around the uptake and the free quantity of ribose. 

Numerous techniques have been used to study ribose, including mass spectrometry, magnetic resonance spectroscopy, and Förster resonance energy transfer (FRET). Our research focused on this last technique. In short, FRET is the energy transfer between a donor and an acceptor fluorescent probe. Over a specific range of distances, excitation of the donor should emit energy that is able to excite the acceptor. This technique was chosen for its biocompatibility. There exists a protein called ribose binding protein (RBP), which exists in a class of proteins called periplasmic binding proteins (PBPs). PBPs change conformation upon binding to their target ligand. For RBP, the protein changes from open conformation to closed conformation upon binding to ribose. This is where a FRET sensor comes in; the RBP can be genetically modified to include two fluorescent proteins (FPs). Based on the folding and conformational changes of RBP, FRET signals can be detected to monitor ribose uptake and concentration. The need for this monitoring and technology comes from literature that suggests ribose is used for cellular pathways associated with cancer cells. Thus, a biosensor for use in living cells is needed.

A plasmid construct with a modified RBP containing green and red FPs (GR-RBP) was transformed into Escherichia coli strain BL21(DE3), and the bacterial cells were cultured and allowed to overexpress the GR-RBP. After expression of the protein and lysing of the cells, the protein had to be purified from the lysate. Experimentation was centered on attempting to purify the protein with the assistance of Mg2+ to remove chaperone proteins from the GR-RBP, which assist in the proper folding of the protein by binding to the protein. Literature showed that Mg2+ could release chaperones, and so MgCl2 was included during the cell culturing and protein overexpression step.

The cell lysate went through two purification steps via fast protein liquid chromatography (FPLC): metal affinity chromatography and size exclusion chromatography (SEC). After purification, the samples collected were run on an SDS-PAGE gel and imaged with fluorescent imaging. The FPLC and gel data showed the protein existing in multiple folding states, evident by multiple peaks on SEC (i.e., multiple sizes of the protein were present) and multiple bands on the gel. The sample that contained GR-RBP was analyzed with fluorometry to investigate if it was functional and able to show FRET activity. Both FPs were present, but the acceptor would not excite when the donor was excited. From this data, it can be concluded that  Mg2+ alone does not allow GR-RBP to be properly expressed and purified.

Future work will focus on redesigning the GR-RBP with a new placement of the donor FP (red) and a linker to give space for RBP to fold.