Team:Washington/Future Plans


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Future Plans

Examination of more proteins

Like any new up and coming technique, the Degron system will require further testing with a larger variety of well studied mutant variants of a single protein, as well as a larger number of well studied proteins in general, before the system can truly be accepted. This is especially important because the proteins we have used to test the Degron system are mostly alpha-helical. In order to make sure that our system is generalizable to all protein topologies, we must test to see if we can obtain similar results with proteins of other topologies.

Our current plans for the future are to test a protein 33RM2 and its less stable variant, 33CL1, both of which bind to PD-1 (a negative t-cell regulator that prevents the recognition of tumorous cells by the immune system). Since the stability of both of these proteins are known and have been verified using other techniques, such as thermal melts, they are very suitable candidates for testing our Degron system.

Further evolving more stable variants of existing proteins

Throughout this past summer, our team has been evolving more stable variants of BINDI through error-prone PCR. Going forwards, we will continue this process and will continually analyze the mutants with flow cytometry and select cells that exhibit higher fluorescence with FACS.

Recommendations for Degron system use

We offer a couple recommendations for those who would like to utilize our novel method of protein stabilization:
1. Use only Deg0, Deg1, and Deg2 for your stability testing. We offer this recommendation because Deg3 and Deg4 are just a parallel version of Deg2 and Deg1, respectively. That is, making all five constructs would be redundant as, theoretically, Deg1 and Deg4 (or Deg2 and Deg3) should have a similar stability, and hence GFP production. In order to minimize extra work, we recommend you utilize only Deg0, Deg1, and Deg2.
2. Utilize E. coli as your test organism, rather than S. cervisiae . Generally, E. coli is easier to work with as it has a faster growth and is very commonly used in laboratories.

Future Applications

Protein engineering is highly important in many areas. Our system allows a generalizable and high throughput method of engineering proteins, and is also less time consuming than current stabilization methods. Engineered proteins selected through this method could be produced in bacteria and aid in the development of thermostable, de novo protein therapeutics.