Part:BBa_K5316012

bfVFP

Branchiostoma floridae Violet Fluorescent Protein (bfVFP) is a photostable and pH-insensitive protein with an emission wavelength of 430 nm, offering stable fluorescence for visualizing biological processes in various environments. This protein was engineered and characterized by Roldán-Salgado et al., who expanded the toolkit of violet-emitting fluorescent proteins.

While most GFP-like proteins rely on chromophores containing a 4-(p-hydroxybenzylidene)-5-imidazolinone moiety, bfVFP, is unique in its structure. Its chromophore lacks the imidazolinone ring and instead consists of a single oxidized tyrosine, flanked by glycine and alanine, making it the simplest chromophore among fluorescent proteins. This violet-emitting protein was generated by two synergistic mutations (S148H and C166I) in the yellow fluorescent protein lanFP10A2.

The structure of the bfVFP chromophore was confirmed using X-ray crystallography and high-resolution mass spectrometry by Roldán-Salgado et al. With excitation and emission wavelengths of 323 nm and 430 nm, a quantum yield of 0.33, and an extinction coefficient of 14,300 M−1 cm−1, bfVFP is highly suitable for multicolor imaging and Förster resonance energy transfer (FRET) when paired with blue or cyan fluorescent proteins.

(All of the information provided in this description has been adapted from Roldán-Salgado et al.’s paper: “A novel violet fluorescent protein contains a unique oxidized tyrosine as the simplest chromophore ever reported in fluorescent proteins”).

Design considerations

The original sequence suffered two different sets of modifications.

The iGEM Barcelona-UB team of 2024 reverse-translated the original protein sequence from Roldán-Salgado et al. to match the most frequently used codons in Homo sapiens, improving its compatibility with human cells for better expression.

Reverse transcription was conducted using the Sequence Manipulation Suite from bioinformatics.org (https://www.bioinformatics.org/sms2/rev_trans.html), using the codon frequencies provided by Kazusa (https://www.kazusa.or.jp/codon/cgi-bin/showcodon.cgi?species=9606&aa=1&style=GCG)

After this codon optimisation, a SapI illegal site appeared at the position 144-150. The AGC nucleotides (148-150), encoding a Serine, were changed to TCC, also encoding a Serine. The frequency of the AGC codon in human cells is of 0.24, whereas that of the TCC codon is of 0.22. Therefore, the change is minimal.

Sources

Sequence sourced from Protein Science. 2022;31:688–700: https://onlinelibrary.wiley.com/doi/10.1002/pro.4265