Part:BBa_K2647001
mRFP linked to spider silk repeats and keratin binding domain
This part comprises of chromoprotein monomeric RFP linked to 6 repetitive sequences of spider dragline silk ADF-3 from European garden spider Araneus diadematus and keratin binding domain C from human desmoplakin. Silk repeats are flanked with 23 amino acid long naturally occurring N- and C-terminal linkers, derived from major ampullate spidroin 1 from Euprosthenops australis. Linkers act to prevent the disordered structure of the silk repeats to disturb the folding of the chromoprotein and the binding domain.
Contents:
1. Usage and Biology
2 Large Scale Production
2.1 Cultivation and Induction
2.2 Cell Lysis and Purification
3. Concentration process
4. Keratin binding
1. Usage and Biology
Spider dragline silk is extremely strong and elastic making it tougher than most of the natural or manmade fibers. This construct code protein which could be used for forming pre-dyed silk fibers or to be used as a coating for keratin based materials.
KBD is one of the three keratin binding domains from human desmoplakin which have been shown to bind to epithelial cytokeratins and vimentin. [1] the structure of cytokeratin and for example hair keratin is similar which is why KBD of desmoplakin is expected to bind also to hair. [2] In our experiments we tested how the chromoprotein with KBD would bind to human hair.
Silk is stable in higher temperatures and so are mRFP and KBD based on our modelling results which enables this construct to be lysed using heat which can lower the production costs.
2 Large Scale Production
2.1 Cultivation and Induction
In order to get enough material for our tests we started production in large scale. Protein expression was started by inoculating 1-2 colonies of expression strain cells which were transformed with plasmids containing our gene of interest to 5ml of LB medium with 50ug/ml Kanamycin and incubated at + 37°C with shaking over night. This starting cultivation was then inoculated to 45ml of LB medium with 50ug/ml Kanamycin and incubated at +37°C with shaking for ~7 hours until the OD600 value was between 0.6-0.8. The whole 50ml was then inoculated to 450ml of LB medium with 50ug/ml Kanamycin in 1l and induced at the same time with final concentration of 0.5mM of IPTG. The flask was sealed with air permeable membrane and inoculated at +30°C with gentle shaking over the weekend.
2.2 Cell Lysis and Purification
Cells were harvested next day by transferring the culture to sorvall centrifuge bottle and centrifuged at 10 000 for 30 minutes. Supernatant was discarded and the pellet was resuspended in 10ml of lysing mix (DNAse1, Lysozyme, 5mM magnesium chloride, 50mM Trish-HCL pH 7.4, protease inhibitor) for 1g of cell pellet and incubated at room temperature for 30 minutes. Cells were sheared using sonification after which the sample was incubated at +70°C waterbath for 30 minutes. Because the silk is unstructured it does not denaturate and PrancerPurple as well as KBD, according to our modelling results, are heat resistant we can denature all the other soluble proteins with heat and we are left with our protein in the supernatant when centrifuged at 10 000 for 90 minutes.
3. Concentration process
The protein was concentrated with Econo-Pac® 10DG Desalting Prepacked Gravity Flow Columns. In principle, when the silk protein is concentrated silk repeats interact with each other forming a fiber. We tried to make fibers by inserting tweezers in our concentrated solution and carefully opening them. We added a drying step by using hot air, to further concentrate the solution.
4. Keratin binding
Samples were desalted using Econo-pac columns and varying concentrations of sample and keratin were mixed: in weight:weight ratios of 1:1, 1:10, 1:100. The mixed samples were incubated for 30 minutes in room temperature after which they were centrifuged for 10 minutes at 4 000g and samples for SDS-PAGE were prepared from supernatant. By centrifuging the solutions all the proteins bound to keratin will be pelleted in the bottom of the tube and everything else that did not bind are present in the supernatant. If there is not a band the size of the protein studied present in the gel, it suggests that protein has bound to keratin.
We identified the band on sample 5 at ~80kDa to be our protein of interest. We analysed that KBD might have bound to keratin in higher keratin concentration (20mg) which can be seen as a lack of band at ~80kDa on sample 1 (figure ). In lower concentration there did not seem to be any binding which can be seen as bands on the samples 3 and 4. However, this might be a result of there being too much protein compared to keratin making the keratin saturated and there is non-bound protein left in the solution.
References
Sequence and Features
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