Optimization of the expression conditions of fluorescently labeled α-synuclein in Escherichia coli by response surface methodology and proteolysis by tobacco etch virus protease
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Alpha-synuclein is an intrinsically disordered protein prone to aggregation and it is involved in the formation of brain tissue amyloids in patients with Parkinson's and other neurodegenerative diseases. By fluorescently labeling recombinantly expressed proteins, the processes of expression and purification of the same can be easily visually monitored. In a previous work, we have constructed three plasmids for the expression of α-synuclein fused C-terminally to His-tagged mCerulean3 through a polyasparagine linker and tobacco etch virus (TEV) proteolytic site. Here we have transformed these vectors into Escherichia coli BL21(DE3) and BL21(DE3)pLysS and optimized the expression conditions by response surface methodology (temperature, time after induction, concentration of induction reagent) of the chimeric proteins coded by vectors. Expression of the chimeric protein was tested at optimal conditions in different media and terrific broth gave the highest yield. The obtained chimeric prot...ein was purified by immobilized metal-affinity chromatography (IMAC), yielding ~29 mg of chimeric protein per liter of medium and was shown to be successfully proteolyzed by TEV protease, giving a fragment of α-synuclein of expected electrophoretic mobility. After another round of IMAC, the obtained α-synuclein of native sequence was mainly found to be in the flow-through.
Кључне речи:
Recombinant technology / Optimization / Response surface methodology / alpha-synuclein / Fluorescent proteinИзвор:
8th Conference of Young Chemists of Serbia, 29th October 2022, Belgrade, 2022, 10-10Издавач:
- Serbian Chemical Society
- Serbian Young Chemists’ Club
Финансирање / пројекти:
- LEAPSyn-SCI - Late Embryogenesis Abundant Proteins: Structural Characterisation and Interaction With Α-Synuclein (RS-ScienceFundRS-Promis-6039663)
- Министарство науке, технолошког развоја и иновација Републике Србије, институционално финансирање - 200168 (Универзитет у Београду, Хемијски факултет) (RS-MESTD-inst-2020-200168)
Напомена:
- Book of Abstracts
Институција/група
Institut za molekularnu genetiku i genetičko inženjerstvoTY - CONF AU - Savić, Aleksa AU - Vidović, Marija AU - Radosavljević, Jelena PY - 2022 UR - https://imagine.imgge.bg.ac.rs/handle/123456789/1831 AB - Alpha-synuclein is an intrinsically disordered protein prone to aggregation and it is involved in the formation of brain tissue amyloids in patients with Parkinson's and other neurodegenerative diseases. By fluorescently labeling recombinantly expressed proteins, the processes of expression and purification of the same can be easily visually monitored. In a previous work, we have constructed three plasmids for the expression of α-synuclein fused C-terminally to His-tagged mCerulean3 through a polyasparagine linker and tobacco etch virus (TEV) proteolytic site. Here we have transformed these vectors into Escherichia coli BL21(DE3) and BL21(DE3)pLysS and optimized the expression conditions by response surface methodology (temperature, time after induction, concentration of induction reagent) of the chimeric proteins coded by vectors. Expression of the chimeric protein was tested at optimal conditions in different media and terrific broth gave the highest yield. The obtained chimeric protein was purified by immobilized metal-affinity chromatography (IMAC), yielding ~29 mg of chimeric protein per liter of medium and was shown to be successfully proteolyzed by TEV protease, giving a fragment of α-synuclein of expected electrophoretic mobility. After another round of IMAC, the obtained α-synuclein of native sequence was mainly found to be in the flow-through. PB - Serbian Chemical Society PB - Serbian Young Chemists’ Club C3 - 8th Conference of Young Chemists of Serbia, 29th October 2022, Belgrade T1 - Optimization of the expression conditions of fluorescently labeled α-synuclein in Escherichia coli by response surface methodology and proteolysis by tobacco etch virus protease EP - 10 SP - 10 UR - https://hdl.handle.net/21.15107/rcub_imagine_1831 ER -
@conference{ author = "Savić, Aleksa and Vidović, Marija and Radosavljević, Jelena", year = "2022", abstract = "Alpha-synuclein is an intrinsically disordered protein prone to aggregation and it is involved in the formation of brain tissue amyloids in patients with Parkinson's and other neurodegenerative diseases. By fluorescently labeling recombinantly expressed proteins, the processes of expression and purification of the same can be easily visually monitored. In a previous work, we have constructed three plasmids for the expression of α-synuclein fused C-terminally to His-tagged mCerulean3 through a polyasparagine linker and tobacco etch virus (TEV) proteolytic site. Here we have transformed these vectors into Escherichia coli BL21(DE3) and BL21(DE3)pLysS and optimized the expression conditions by response surface methodology (temperature, time after induction, concentration of induction reagent) of the chimeric proteins coded by vectors. Expression of the chimeric protein was tested at optimal conditions in different media and terrific broth gave the highest yield. The obtained chimeric protein was purified by immobilized metal-affinity chromatography (IMAC), yielding ~29 mg of chimeric protein per liter of medium and was shown to be successfully proteolyzed by TEV protease, giving a fragment of α-synuclein of expected electrophoretic mobility. After another round of IMAC, the obtained α-synuclein of native sequence was mainly found to be in the flow-through.", publisher = "Serbian Chemical Society, Serbian Young Chemists’ Club", journal = "8th Conference of Young Chemists of Serbia, 29th October 2022, Belgrade", title = "Optimization of the expression conditions of fluorescently labeled α-synuclein in Escherichia coli by response surface methodology and proteolysis by tobacco etch virus protease", pages = "10-10", url = "https://hdl.handle.net/21.15107/rcub_imagine_1831" }
Savić, A., Vidović, M.,& Radosavljević, J.. (2022). Optimization of the expression conditions of fluorescently labeled α-synuclein in Escherichia coli by response surface methodology and proteolysis by tobacco etch virus protease. in 8th Conference of Young Chemists of Serbia, 29th October 2022, Belgrade Serbian Chemical Society., 10-10. https://hdl.handle.net/21.15107/rcub_imagine_1831
Savić A, Vidović M, Radosavljević J. Optimization of the expression conditions of fluorescently labeled α-synuclein in Escherichia coli by response surface methodology and proteolysis by tobacco etch virus protease. in 8th Conference of Young Chemists of Serbia, 29th October 2022, Belgrade. 2022;:10-10. https://hdl.handle.net/21.15107/rcub_imagine_1831 .
Savić, Aleksa, Vidović, Marija, Radosavljević, Jelena, "Optimization of the expression conditions of fluorescently labeled α-synuclein in Escherichia coli by response surface methodology and proteolysis by tobacco etch virus protease" in 8th Conference of Young Chemists of Serbia, 29th October 2022, Belgrade (2022):10-10, https://hdl.handle.net/21.15107/rcub_imagine_1831 .