Team:Linkoping Sweden/Protein

The Protein

The protein is a composition of the biobricks BBa_E1010 (red fluorescent protein, RFP) and BBa_K525998 (promotor T7 and RBS) which are inserted together with Epitope 2 from Ara h 1 into a psb1c3-vector.

When digesting and ligating the biobricks in order to obtain the desired sequence, a stop codon appeared between the biobricks, this problem was solved using single point mutagenesis where the stop codon was exchanged with leucine. Leucine is a small amino acid with an unpolar side chain and will therefore have a very small impact on the protein.

To avoid misfolding of the protein, a linker composed of the amino acids glycine and serine is used to separate the epitope from RFP. Glycine is a small amino acid and provides flexibility1 to the linker while serine adds stability2.

GS-linker is used to separate the epitope sequence from RFP.

Epitope 2 was chosen due to its location on the surface of the Ara h 1 allowing the antibodies to recognize the sequence in it’s native form unlike epitopes located on the inside of Ara h 1 which can only be recognized when the full length protein has been denatured.

The antibodies which the protein construct is supposed to bind to are monoclonal and specific for epitope 2. These antibodies are conjugated with three to six fluorescein isothiocyanate (FITC) molecules which absorbs light at 495 nm and emits at 519 nm. If RFP is close enough it can absorb the light emitted by FITC and emit light at a higher wavelength; 607 nm. This phenomenon is called Förster resonance energy transfer (FRET).

Amino acid sequence

The epitopes amino acid sequence and an explanation of its building blocks:

MHHHHHHSSGVDLGTENLYFQSMQEPDDLKQKAGSGSGSGSGSYLMASSEDVIKEFMRFKVRMEGSVNGHEFEIEGEGEGRPYEGTQTAKLKVTKGGPLPFAWDILSPQFQYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASTERMYPEDGALKGEIKMRLKLKDGGHYDAEVKTTYMAKKPVQLPGAYKTDIKLDITSHNEDYTIVEQYERAEGRHSTGA**R**C*CRSLLVAAAA

HHHHHH

The Histidine tag which is used in order to simplify the purification of the protein when loaded onto a nickel column.

ENLYFQS

Tev protease recognision site which cuts the His-tag.

QEPDDLKQKA

Epitope 2 from Ara h 1.

GSGSGSGSGS

GS-linker used to separate epitope 2 and RFP to avoid misfolding

*

Stop codon

The rest of the sequence is RFP from the biobrick E1010. This creates the protein complex of epitope and RFP, shown i Figure 1.

Figure 1. Prediction of the three-dimensional structure model of the full protein complex obtained from I-Tasser.3

References

  • 1. Ryan Trinh, Brian Gurbaxani, Sherie L Morrison, Manouchehr Seyfzadeh (2004). Optimization of codon pair use within the (GGGGS)3 linker sequence results in enhanced protein expression
  • 2. Clifford R. Robinson, Robert T. Sauer (1998). Optimizing the stability of single-chain proteins by linker length and composition mutagenesis
  • 3. http://zhanglab.ccmb.med.umich.edu/I-TASSER/output/S240383/w25vap/