To determine the most conserved substitutions for free cysteines in the binding proteins, amino acid sequences were inserted into Phyre2, an online server for protein homology which runs an intensive BLAST search, giving a sequence homology output in FASTA format. Sequences for phosphate binding protein, potassium binding protein, glucose/galactose binding protein, alpha-klotho, and the parathyroid hormone receptor (N-termini) were inserted.
Compared to other binding proteins, alpha-klotho had the most free cysteines, requiring the most substitutions. Interestingly, glucose, potassium, and the parathyroid hormone receptor N-termini had no free cysteines, so only one substitution was made – inserting a cysteine to later bind to a coil. Both glucose and potassium binding proteins had their C-termini trimmed by 23 and 8 amino acids, respectively, a recommendation found in literature. To read more about how we mutated, trimmed, and identified residues for cysteine type immobilization refer to our protocol section.
After substitutions took place, mNeonGreen and mCherry were added to the N- and C- terminis of our binding proteins respectively, to allow for fluorescent detection of biomarkers. With phosphate binding protein, a 2 amino acid glycine-threonine linker was inserted between the fluorophores and binding protein, a recommendation from literature. It is worth noting that the parathyroid hormone receptor and alpha-klotho only have a N- termini fluorophore attached. This is because they are involved in competition type assays, with either PTH or FGF23 tagged with the partner fluorophore. More information on this may be found in our contributions or protocol pages.
Team:Queens Canada/Results