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Title: Engineering thermostable protein nanocompartments for protein production and enhanced enzyme activity
Author: Zarazúa Arvizu, Efraín
ISNI:       0000 0004 8509 4930
Awarding Body: University of Edinburgh
Current Institution: University of Edinburgh
Date of Award: 2020
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The bacterial nanocompartments (encapsulins), produced by some species of bacteria and archaea, possess characteristics that can potentially overcome current challenges related to recombinant production of proteins. Encapsulins are simple icosahedral protein nanocages that encapsulate single enzyme species to protect the cell from oxidative damage. They are beneficial to cells because they increase the local concentration of enzymes, discriminate access of molecules, ease substrate transfer and enclose toxic products. In this study we heterologously encapsulated the fluorescent protein PhiLOV into two different encapsulins produced by Rhodospirillum rubrum (Rru Enc) and Rhodococcus opacus (Rho Enc). The short C-terminal peptide from the native cargo proteins found in R. rubrum and R. opacus encapsulins (Ferritin-like protein and Dye peroxidase, respectively) was appended to the C-terminal region of PhiLOV (PhiLOV_ES) to direct the protein into the encapsulin shell. All the constructs were recombinantly expressed in E. coli BL21(DE3) cells and purified after cell lysis, using Ion-Exchange and Size Exclusion Chromatography. Both encapsulins were copurified with the fluorescent cargo PhiLOV_ES protein. The purified encapsulins were analysed through Transmission Electron Microscopy (TEM) to verify the formation of self-assembled icosahedral shells. In both samples, shells of approximately 20-22 nm of diameter were observed. The accurate masses of Rru Enc, Rho Enc and PhiLOV_ES monomers obtained by Liquid Chromatography - Mass spectrometry analysis was consistent with the masses of the proteins calculated in silico. To demonstrated that the fluorescent cargo protein was located inside the capsid, PhiLOV_ES proteins were tagged with a N-terminal degradation signal (RepA). We hypothesised that encapsulated cells should avoid the proteolysis process by using the encapsulins as a protective shell. Cells expressing the tagged PhiLOV_ES (RepA_PhiLOV_ES) without the encapsulin did not show fluorescence after 4 hours of induction. Conversely, cells coexpressing the RepA_PhiLOV_ES and the encapsulin showed high fluorescent levels. Finally, we developed a nano-encapsulation platform that includes a substantial genetic toolbox library, a robust purification expression and purification process that allows to target heterologous proteins within encapsulins and might be useful to develop novel functional encapsulins with potential industrial applications.
Supervisor: Horsfall, Louise ; Bramham, Janice Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
Keywords: nanocompartments ; encapsulins ; synthetic biology