Part:BBa_K3237016
His-mRFP1-Proinsulin with Furin cut sites Codon Optimized for E.coli
This part is a fusion protein where a pro-insulin derived part is fused to mRFP1. The mRFP1 provides a way to detect expression in vivo . Linkers in between the fused proteins ensure proper folding and prevent steric hindrance. As well, a furin cut site (a human protein that is a sequence specific protease)[1] was added to remove the mRFP1. This part is an improvement on the part from Tsinghua 2014 BBa_K1328003 which also has replaced the natural protease sites in insulin with furin cut sites. Finally, there was a his tag added onto the RFP to allow for affinity purification of the protein.
In summary, the protein is expressed as a RFP-proinsulin fusion. The protein is then purified by affinity purification. After purification the protein can be treated with furin protein. At this point RFP and the insulin is separated and the insulin is processed into its bioactive form. For more information on our design please go to our wiki Lethbridge iGEM 2019. For our data reuslts please go to our composite parts page BBa_K3237016.
Although the following data was primarily done in pUC57 we have also been able to clone this part into Pet28a(+). Our sequence confirmation file can be found here.
Protein Purification
Figure 1: A 10% SDS-PAGE of the nickel affinity purification using the AKTA START system of the mRFP-proinsulin protein. Our protein is seen at between the 25kDa and 35kDa mark. This is lower than expected and is likely due to protease degredation.
Figure 2: A chromatogram of the proinsulin affinity purification. The blue line represents the A280 reads that correspond to our protein and the orange line corresponds to the amount of elution buffer (high imidazole content) used.
Furin Cleavage Assay
Although several attempts were made, we were unable to cut the proinsulin using the protease. 3.43, 2.58 and 2.38kDa bands were expected to be seen but as compared to the control, there was not change to the sample.
Figure 1: A 16.5% Tricine PAGE with mRFP1-Proinsulin and mRFP1-SCI57 treated with Furin Protease at 25 degrees Celsius for 16 hours. The gel was run at 100V for 10 minutes, then 180V for 60 minutes and stained with coomassie. Lane 1= 10-240kDA marker (Biobasic), lane 2= mRFP1-Proinsulin 10 units Furin, Lane 3= mRFP1-Proinsulin 20 units Furin, Lane 4= mRFP1-Proinsulin untreated, Lane 5= mRFP1-SCI57 10 units Furin, Lane 6= mRFP1-SCI57 20units Furin, Lane 7=mRFP1-SCi57 untreated.
To determine if there was potential furin binding hinderance we decided to model the cutting of the mRFP1-proinsulin construct. According to the I-Tasser program there is no reason why binding may not occur.
Figure 1: 3D structural model of 6xHIS-RFP-Proinsulin-CPP. The N-terminal HIS tag is shown in blue and the C-terminal CPP is shown in red. The characteristic beta-barrel scaffold of RFP is clearly evident and appropriately folded.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 7
Illegal NheI site found at 30 - 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 649
Illegal AgeI site found at 761 - 1000COMPATIBLE WITH RFC[1000]
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