Through iterative cycles of computational design, testing, and optimization, the team successfully created the A6T7 Chimera PFAS-degrading enzyme and improved protein expression techniques despite initial challenges. The first design success we detail in the main page of our part, https://parts.igem.org/Part:BBa_K5035003. We will use the remainder of the page to describe an early challenge we overcame with protein expression.
Pellet of insoluble aggregate of T7RdhA formed at the bottom of the tube
Denatured T7RdhA was successfully purified from a cell lysate solution, as shown in this protein electrophoresis gel.
To fix this issue, the team devised two potential strategies: infusing the expression solution with the cofactors so that as the protein is translated, the cofactors integrate into the structure of the protein or express T7RdhA without cofactors and then denature the T7RdhA inclusion bodies and renature them with cofactors present to reconstitute them into their structure. We decided to attempt both strategies simultaneously. The first strategy did result in the absence of the inclusion body pellet; however, when purified, only a faint band or no band at all at the correct size was observed, leading us to conclude that this method only resulted in minimal yield. The second method, we dubbed the Nakamura method after the primary author of the paper that details it, resulted in a much clearer band at the correct size, the downside being the complexity and lengthiness of the protocol.
The Nakamura protocol results in a higher yield of refolded enzymes than previous folding methods.
With this success, we went on to express O68252, A6T7 Chimera, A0A7C3HHU1, A0A2E5NSL8, and A0A419DFF0 and send these purified enzyme solutions off-site to be tested against a PFAS substrate.
See our Results, Contribution, Notebook, and Project Description pages for details of the iterative process, and links to our parts.
