Part:BBa_K3809011
mRFP with Linker, His Tag and Signal Peptide
This sequence codes for protein reporter mRFP. It was modified in order to express the complete sequence of mRFP, with some additions. The sequence also includes a 6X Histidine Tag (for its purification), a linker sequence of 4 glycines, 1 serine and 1 cistein (for its immobilization) and a signal peptide sequence (NPS4). The purose of expressing this protein is to analyze its folding and correct immobilization.
For the purification of mRFP1, we added a 6X His Tag and for the immobilization of the protein in a piezoelectric sensor, we added a linker which includes a sequence of 4 glycines, 1 serine and 1 cistein. The addition of the cistein is very important since the immobilization method we chose is based on the formation of a disulfide bond. Furthermore, we chose to mantain the DNA binding domain because it has a region rich in cisteins. Finally, we added a signal peptide sequence NPS4 (which comes from BBa_K3606042), in order to guarantee the secretion of our protein and make it easier for us to purify.
To verify that mRFP1 would fold correctly and there would be no steric obstacle, we simulated the protein with its corresponding additions using the tool Robetta (https://robetta.bakerlab.org/). When we recieved the model, we made an structural alignment using the natural protein as reference. Therefore, we concluded that the addition of the Linker, His Tag and Signal Peptide were not interfering with the correct folding of mRFP1. Furthermore, we proved that the His Tag and the linker were exposed, so we could use them for the purposed we established previously.
This results were important since it also proved the functionality of ESR1 (BBa_K38090109).
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 48
Illegal AgeI site found at 651
Illegal AgeI site found at 763 - 1000COMPATIBLE WITH RFC[1000]
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