Team:Aachen/Proof
Proof of Concept
Photocaging of Subtilisin E
To prove the principle of our project idea, we performed a site-directed mutagenesis on each of our targeted sites to simulate the integration of a photo-labile, non-canonical amino acid. At first, we exchanged serine221 in the active site of subtilisin E against tyrosine. In addition to this, we substituted tyrosine77 in the pro-peptide cleavage site against tryptophan.
Then, the cells with a modified version of the expression system for subtilisin E in E. coli that had been proven to work beforehand were streaked on skim milk agar plates containing the inducer IPTG and the needed antibiotics.
Neither the empty backbone nor the expression system with the modified catalytic triade seems to cause a proteolytic activity. A clearance of the skim milk plates and therefore a proteolytic activity can only be observed for the native protease (as it has been demostrated before) and the Y77W-mutated enzyme.
Through this experiment, we are now able to prove that serine is essential for the proteolytic activity of the protease and that exchanging it would inactivate the enzyme.
As seen on the pictures above, a clearance had occurred for the cells modified to express subtilisin with tryptophan instead of tyrosine77. As a result, a proteolytic activity can be assumed. Contrary to our former beliefs, it can now be deduced that exchanging tyrosine doesn’t result in a change of activity. Consequently, tyrosine in the pro-peptide cleavage site is not essential for the activity of subtilisin E.
Conclusion
Unfortunately, tyrosine77 seems to be not essential for the proteolytic activity. Thus, exchanging tyrosine against ONB-tyrosine did not influence the activity of the enzyme.
On the other hand, we are able to proof that exchanging serine221 in the active site will result in a loss of activity. Hence, we have demonstrated that substituting serine against a photo-labile, non-canonical amino acid like DMNBS will inactivate subtilisin E. Thus, we can now provide a valid proof of the principle of our project.
Dark Bench:
In order to prove that our dark bench is light proof, we needed to characterize our optical windows and safelight. As it is already known that the emission of red LED is well below 500 nm, we have only analyzed the absorption spectrum of our windows specimen. From the graph below, we can confirm that, in the wavelength below 500 nm, the specimen blocks about 99% of incident radiation in UV-A region and 99% in blue region
Conclusion
In short, we demonstrated that building a device which is affordable, convenient, and provides a light proof workspace, is possible.
LIPs-Stick
To validate that our UV-exposure device called LIPs-Stick, activates the caged protease, we studied the photo-cleavage reaction of photo-caged amino acid by visible spectrophotometry and found that our device can effectively activate the caged amino acid. From the graph below, we can see not only the formation of cleavage products but also the amount of the cleavage product depends on UV intensity and exposure time.
Conclusion
To sum up, we proved that an inexpensive and compact tool can be built, which can activate photo-caged amino acid.
