Part:BBa_K3237018
His-mRFP1-SCI57 with furin cut sites codon optimized for E.coli
The SCI57 insulin protein is a synthetic insulin that is a single chain analog that found to be ultra stable [1]. being that it is very stable, we have chosen it as a candidate for studies in expressing and delivering insulin in microalgae. Along with the coding sequence for this single chain insulin, we have also fused it to mRFP1 also known as BBa_E1010. in between this fusion is a furin cut site [2] and a linker in order to remove the mRFP once in contact with the human protease. The other component added is a hexahistidine tag added to the N-terminus of the construct for purification purposes. Although our project deals with expression in microalgae, this construct was created for comparative expression studies and was therefore codon optimized for E.coli .
Although most of our work was done in pUC57, we were able to clone our this part into Pet28a(+). Our confirmation of sequencing file can be found here.
Nickel Affinity Purification
Figure 1: 10% SDS-PAGE gel of the Nickel affinity Purification of mRFP1-SCI57 protein using the ACTA START system.
Figure 2: A chromatogram of mRFP1-SCI57 nickel affinity purification. The blue line represents the A280 reads which are the proteins coming off the column. The orange line is the amount of Elution buffer (high imadazole content) used during the purification.
Furin cleavage Assay
Although several attempts were made, we were unable to cut the proinsulin using the protease. A band at 6.08kDa was expected 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.
MicraScale Thermophoresis Assay
As we were unable to cut the protein with the furin protease (perhaps due to deactivated protein or issues with the furin being able to reach the cut site in the protein) and due to time constraints we decided to run a MicroScale thermophoresis assay with the unprocessed SCi57 sample.
Figure 1: A time scale graph showing the MicroScale Thermophoresis assay indicating protein-protein interactions between the mRFP1-SCI57 protein and the insulin receptor. The blue data indicates fluorescence reads with just the mRFP1-SCI57 construct at 500nM concentration. The green data represents the samples containing the mRFP1-SCI57 and insulin receptor at 500nM concentration and ~17,000nM receptor respectively. The increase in fluorescence reads with the mRFP1-SCi57 + insulin receptor protein indicates a binding event between the two proteins.
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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