Densitometric analysis of protein profiles as a tool for DoE decision making for recombinant protein production
Конференцијски прилог (Објављена верзија)
Метаподаци
Приказ свих података о документуАпстракт
The optimization of expression conditions is a tedious, but necessary procedure for the
successful efficient production of recombinant proteins. Design of experiment (DoE) saves
time and resources by using statistics to carefully select only a few experimental points
(expression conditions) to obtain information about the whole tested range of varied
conditions, unlike the most commonly used one-factor-at-a-time (OFAT) method, which
requires full imput space screening for optimization. Here, we have optimized the
expression conditions of His 6 and His8-mCerulean3-TEV-α-synuclein production from the
the pDUET vector, as well as the His 6-tagged protein from the pET-20b vector and
expressed into the periplasm. The proteins for which optimization was conducted were
expressed in Escherichia coli BL21(DE3) and BL21(DE3)pLysS. We have used DoE to
plan adequate experimental points for optimization according to a Box-Behnken design,
with IPTG concentration, temperature and time vari...ation. After collecting the cultures at
given experimental points, they were analyzed by SDS-PAGE and densitometry, then
modelled and statistically analysed. The importance of correct sample preparation and gel
loads for densitometric analysis is evident according to the obtained results. The largest
expression level was obtained from the His 8-tagged protein coded by the pDUET vector in
E. coli BL21(DE3). We have also used densitometry to analyze expression levels in
different culture media.
Кључне речи:
Design of Experiment (DoE) / Box-Behnken design / Densitometry / Expression optimizationИзвор:
Zbornik apstrakata, VI Simpozijum Srpskog udruženja za proteomiku (SePA) “Razvoj i primena novih metoda proteomike”, 2. jun 2023. godine, Kragujevac, 2023, 24-24Издавач:
- Prirodno-matematički fakultet, Univerzitet u Kragujevcu
Финансирање / пројекти:
- LEAPSyn-SCI - Late Embryogenesis Abundant Proteins: Structural Characterisation and Interaction With Α-Synuclein (RS-ScienceFundRS-Promis-6039663)
Напомена:
Повезане информације:
Институција/група
Inovacioni centar / Innovation CentreTY - CONF AU - Savić, Aleksa AU - Radosavljević, Jelena PY - 2023 UR - http://cherry.chem.bg.ac.rs/handle/123456789/6180 AB - The optimization of expression conditions is a tedious, but necessary procedure for the successful efficient production of recombinant proteins. Design of experiment (DoE) saves time and resources by using statistics to carefully select only a few experimental points (expression conditions) to obtain information about the whole tested range of varied conditions, unlike the most commonly used one-factor-at-a-time (OFAT) method, which requires full imput space screening for optimization. Here, we have optimized the expression conditions of His 6 and His8-mCerulean3-TEV-α-synuclein production from the the pDUET vector, as well as the His 6-tagged protein from the pET-20b vector and expressed into the periplasm. The proteins for which optimization was conducted were expressed in Escherichia coli BL21(DE3) and BL21(DE3)pLysS. We have used DoE to plan adequate experimental points for optimization according to a Box-Behnken design, with IPTG concentration, temperature and time variation. After collecting the cultures at given experimental points, they were analyzed by SDS-PAGE and densitometry, then modelled and statistically analysed. The importance of correct sample preparation and gel loads for densitometric analysis is evident according to the obtained results. The largest expression level was obtained from the His 8-tagged protein coded by the pDUET vector in E. coli BL21(DE3). We have also used densitometry to analyze expression levels in different culture media. PB - Prirodno-matematički fakultet, Univerzitet u Kragujevcu C3 - Zbornik apstrakata, VI Simpozijum Srpskog udruženja za proteomiku (SePA) “Razvoj i primena novih metoda proteomike”, 2. jun 2023. godine, Kragujevac T1 - Densitometric analysis of protein profiles as a tool for DoE decision making for recombinant protein production SP - 24 EP - 24 UR - https://hdl.handle.net/21.15107/rcub_cherry_6180 ER -
@conference{ author = "Savić, Aleksa and Radosavljević, Jelena", year = "2023", abstract = "The optimization of expression conditions is a tedious, but necessary procedure for the successful efficient production of recombinant proteins. Design of experiment (DoE) saves time and resources by using statistics to carefully select only a few experimental points (expression conditions) to obtain information about the whole tested range of varied conditions, unlike the most commonly used one-factor-at-a-time (OFAT) method, which requires full imput space screening for optimization. Here, we have optimized the expression conditions of His 6 and His8-mCerulean3-TEV-α-synuclein production from the the pDUET vector, as well as the His 6-tagged protein from the pET-20b vector and expressed into the periplasm. The proteins for which optimization was conducted were expressed in Escherichia coli BL21(DE3) and BL21(DE3)pLysS. We have used DoE to plan adequate experimental points for optimization according to a Box-Behnken design, with IPTG concentration, temperature and time variation. After collecting the cultures at given experimental points, they were analyzed by SDS-PAGE and densitometry, then modelled and statistically analysed. The importance of correct sample preparation and gel loads for densitometric analysis is evident according to the obtained results. The largest expression level was obtained from the His 8-tagged protein coded by the pDUET vector in E. coli BL21(DE3). We have also used densitometry to analyze expression levels in different culture media.", publisher = "Prirodno-matematički fakultet, Univerzitet u Kragujevcu", journal = "Zbornik apstrakata, VI Simpozijum Srpskog udruženja za proteomiku (SePA) “Razvoj i primena novih metoda proteomike”, 2. jun 2023. godine, Kragujevac", title = "Densitometric analysis of protein profiles as a tool for DoE decision making for recombinant protein production", pages = "24-24", url = "https://hdl.handle.net/21.15107/rcub_cherry_6180" }
Savić, A.,& Radosavljević, J.. (2023). Densitometric analysis of protein profiles as a tool for DoE decision making for recombinant protein production. in Zbornik apstrakata, VI Simpozijum Srpskog udruženja za proteomiku (SePA) “Razvoj i primena novih metoda proteomike”, 2. jun 2023. godine, Kragujevac Prirodno-matematički fakultet, Univerzitet u Kragujevcu., 24-24. https://hdl.handle.net/21.15107/rcub_cherry_6180
Savić A, Radosavljević J. Densitometric analysis of protein profiles as a tool for DoE decision making for recombinant protein production. in Zbornik apstrakata, VI Simpozijum Srpskog udruženja za proteomiku (SePA) “Razvoj i primena novih metoda proteomike”, 2. jun 2023. godine, Kragujevac. 2023;:24-24. https://hdl.handle.net/21.15107/rcub_cherry_6180 .
Savić, Aleksa, Radosavljević, Jelena, "Densitometric analysis of protein profiles as a tool for DoE decision making for recombinant protein production" in Zbornik apstrakata, VI Simpozijum Srpskog udruženja za proteomiku (SePA) “Razvoj i primena novih metoda proteomike”, 2. jun 2023. godine, Kragujevac (2023):24-24, https://hdl.handle.net/21.15107/rcub_cherry_6180 .