Engineering thermostable protein nanocompartments for protein production and enhanced enzyme activity
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Date
06/02/2020Item status
Restricted AccessEmbargo end date
06/02/2022Author
Zarazúa Arvizu, Efraín
Metadata
Abstract
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.