CHARACTERIZATION OF PHOTORECEPTOR-CONTROLLED AGGREGATION AND DISAGGREGATION OF MICROPARTICLE SUSPENSIONS

ECU Author/Contributor (non-ECU co-authors, if there are any, appear on document)

Christopher B Cone (Creator)

Institution

East Carolina University (ECU )

Web Site: http://www.ecu.edu/lib/

Abstract: Phytochromes are a unique classification of photoreceptors used by plants to perceive light for many physiological processes. Phytochrome B (PhyB) possesses two interconvertible forms which can be activated under specific wavelengths of light to bind and unbind an integrating factor PIF3 to carry out gene expression within the nucleus of plant cells. This natural switching mechanism of PhyB to bind and unbind PIF3 provides the potential to create a photo-switchable controlling mechanism as a platform for biotechnological devices. The overall goal of this research was to develop a platform using microparticles coated with PhyB and PIF3 proteins that would interact under the control of light by aggregating and disaggregating based on wavelength, intensity, and duration of light.The development of a microparticle-based platform to immobilize these photoreactive protein counterparts presents many challenges. To express photoresponsive recombinant PhyB protein in E. coli cells, two additional proteins are required to be coexpressed that are toxic to the cells. In addition, expression levels of soluble and active PhyB are very low (around 15%) and must be produced under dark room conditions. Another challenge is to immobilize the PhyB and PIF3 proteins on surfaces that retains their photo-responsive attributes and can interact as part of the device platform. Further challenges are the characterization of these photo-responsive interactions between PhyB and PIF3 to determine the operating conditions and limitations in developing this platform.Truncated versions of PhyB and PIF3 were expressed using benchtop bioreactors and purified using affinity chromatography. The proteins were characterized as freely suspended proteins in solution as well as immobilized on treated polystyrene microparticle surfaces and agarose-coated magnetic particles. The proteins were also characterized by their interactions in which one protein was in suspension while the counterpart protein was immobilized on a microparticle. The interactions between separately-coated protein particles, the essence of the proposed platform, was also investigated. Despite the expected low yields, the purified PhyB protein displayed photo-responsive characteristics based the wavelength of light used (red light, 660 nm for active and far-red light, 730 nm for inactive) as well as dark reversion kinetics (the protein in the active state reverting to its inactive state without photoinduction). However, when immobilized on polystyrene particles or magnetic particles, PhyB seemed to lose its photo-responsiveness, or at least couldn't bind to PIF3 while immobilized and in the active state. Based on gel electrophoresis analysis, the immobilization of truncated PhyB to microparticles seemed to affect the binding of PIF3 in solution when the truncated PhyB was irradiated with red light. On the other hand, depletion assays indicated immobilized PhyB was able to bind and unbind suspended PIF3 in the two photoinduced states. Depletion assays also indicated a more photo-responsive interaction when PIF3 was immobilized on the bead surface while PhyB remained suspended in solution. To test the hypothesis of the platform, aggregation and disaggregation experiments where both proteins were immobilized on separate microparticles were conducted and observed by light microscopy. No aggregation was observed under photoactive red light conditions. The overall results seem to indicate the truncated versions of PhyB and PIF3, while displaying photo-responsive activity in suspension, are inhibited when specifically immobilized on microparticle surfaces.

Additional Information

Publication

Thesis

Language: English

Date: 2023

Subjects

Biohybrid devices;Photoconversion;Photocontrollable devices

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