Electrophoretic assessment of recombinant λ- exonuclease production in different E. coli strains
Конференцијски прилог (Објављена верзија)
Метаподаци
Приказ свих података о документуАпстракт
Viral exonucleases play role in many processes essential for genome ma intenance,
including DNA repair and recombination. Lambda exonuclease (λ -exo), isolated from
lambda bacteriophage, hydrolases double-stranded DNA (dsDNA) in the highly processive
manner in 5’→3’ direction, yielding mononucleotides and single -stranded DNA (ssDNA).
This unique enzymatic properties offer several promising biotechnological applications,
such as highly sensitive quantification of DNA modifications and single -molecule
sequencing. Hence, optimization of the expression conditions is a prerequisite to achieve
high-level production of λ-exo. Here we have tested λ -exo expression in five different E.
coli strains under various temperature regimes in order to establish the optimal conditions
for efficient production of recombinant λ -exo. The N-terminally His -tagged λ-exo was
successfully expressed in E.coli BL21(AI), SHuffle T7, C41(DE3) and C43(DE3) strains in
LB broth. Collected aliquots were ...analysed by SDS-PAGE, followed by CBB staining.
Relative yield of target protein bands was determined by densitometry in total cell lysate, as
well as in soluble and insoluble cytoplasmatic fractions. We identified E.coli BL21(AI),
SHuffle T7 and C41(DE3) as good producers of recombinant λ -exo, and upon scaling up, λ
-exo was purified from crude cell lysates by metal affinity chromatography in satisfactory
yield. Our data suggest that densitometric analysis could serve as a powerful low-cost
screening platform for improving recombinant protein expression strategies.
Кључне речи:
λ-exonuclease / E. coli / DensitometryИзвор:
Zbornik apstrakata, VI Simpozijum Srpskog udruženja za proteomiku (SePA) “Razvoj i primena novih metoda proteomike”, 2. jun 2023. godine, Kragujevac, 2023, 23-23Издавач:
- Prirodno-matematički fakultet, Univerzitet u Kragujevcu
Финансирање / пројекти:
- Министарство науке, технолошког развоја и иновација Републике Србије, институционално финансирање - 200168 (Универзитет у Београду, Хемијски факултет) (RS-MESTD-inst-2020-200168)
- Министарство науке, технолошког развоја и иновација Републике Србије, институционално финансирање - 200026 (Универзитет у Београду, Институт за хемију, технологију и металургију - ИХТМ) (RS-MESTD-inst-2020-200026)
- Министарство науке, технолошког развоја и иновација Републике Србије, институционално финансирање - 200288 (Иновациони центар Хемијског факултета у Београду доо) (RS-MESTD-inst-2020-200288)
Напомена:
- Related to poster: https://cherry.chem.bg.ac.rs/handle/123456789/6206
Повезане информације:
Колекције
Институција/група
Hemijski fakultet / Faculty of ChemistryTY - CONF AU - Stefanović, Marija AU - Savić, Aleksa AU - Božić, Nataša AU - Vujčić, Zoran AU - Radosavljević, Jelena PY - 2023 UR - http://cherry.chem.bg.ac.rs/handle/123456789/6205 AB - Viral exonucleases play role in many processes essential for genome ma intenance, including DNA repair and recombination. Lambda exonuclease (λ -exo), isolated from lambda bacteriophage, hydrolases double-stranded DNA (dsDNA) in the highly processive manner in 5’→3’ direction, yielding mononucleotides and single -stranded DNA (ssDNA). This unique enzymatic properties offer several promising biotechnological applications, such as highly sensitive quantification of DNA modifications and single -molecule sequencing. Hence, optimization of the expression conditions is a prerequisite to achieve high-level production of λ-exo. Here we have tested λ -exo expression in five different E. coli strains under various temperature regimes in order to establish the optimal conditions for efficient production of recombinant λ -exo. The N-terminally His -tagged λ-exo was successfully expressed in E.coli BL21(AI), SHuffle T7, C41(DE3) and C43(DE3) strains in LB broth. Collected aliquots were analysed by SDS-PAGE, followed by CBB staining. Relative yield of target protein bands was determined by densitometry in total cell lysate, as well as in soluble and insoluble cytoplasmatic fractions. We identified E.coli BL21(AI), SHuffle T7 and C41(DE3) as good producers of recombinant λ -exo, and upon scaling up, λ -exo was purified from crude cell lysates by metal affinity chromatography in satisfactory yield. Our data suggest that densitometric analysis could serve as a powerful low-cost screening platform for improving recombinant protein expression strategies. 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 - Electrophoretic assessment of recombinant λ- exonuclease production in different E. coli strains SP - 23 EP - 23 UR - https://hdl.handle.net/21.15107/rcub_cherry_6205 ER -
@conference{ author = "Stefanović, Marija and Savić, Aleksa and Božić, Nataša and Vujčić, Zoran and Radosavljević, Jelena", year = "2023", abstract = "Viral exonucleases play role in many processes essential for genome ma intenance, including DNA repair and recombination. Lambda exonuclease (λ -exo), isolated from lambda bacteriophage, hydrolases double-stranded DNA (dsDNA) in the highly processive manner in 5’→3’ direction, yielding mononucleotides and single -stranded DNA (ssDNA). This unique enzymatic properties offer several promising biotechnological applications, such as highly sensitive quantification of DNA modifications and single -molecule sequencing. Hence, optimization of the expression conditions is a prerequisite to achieve high-level production of λ-exo. Here we have tested λ -exo expression in five different E. coli strains under various temperature regimes in order to establish the optimal conditions for efficient production of recombinant λ -exo. The N-terminally His -tagged λ-exo was successfully expressed in E.coli BL21(AI), SHuffle T7, C41(DE3) and C43(DE3) strains in LB broth. Collected aliquots were analysed by SDS-PAGE, followed by CBB staining. Relative yield of target protein bands was determined by densitometry in total cell lysate, as well as in soluble and insoluble cytoplasmatic fractions. We identified E.coli BL21(AI), SHuffle T7 and C41(DE3) as good producers of recombinant λ -exo, and upon scaling up, λ -exo was purified from crude cell lysates by metal affinity chromatography in satisfactory yield. Our data suggest that densitometric analysis could serve as a powerful low-cost screening platform for improving recombinant protein expression strategies.", 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 = "Electrophoretic assessment of recombinant λ- exonuclease production in different E. coli strains", pages = "23-23", url = "https://hdl.handle.net/21.15107/rcub_cherry_6205" }
Stefanović, M., Savić, A., Božić, N., Vujčić, Z.,& Radosavljević, J.. (2023). Electrophoretic assessment of recombinant λ- exonuclease production in different E. coli strains. 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., 23-23. https://hdl.handle.net/21.15107/rcub_cherry_6205
Stefanović M, Savić A, Božić N, Vujčić Z, Radosavljević J. Electrophoretic assessment of recombinant λ- exonuclease production in different E. coli strains. in Zbornik apstrakata, VI Simpozijum Srpskog udruženja za proteomiku (SePA) “Razvoj i primena novih metoda proteomike”, 2. jun 2023. godine, Kragujevac. 2023;:23-23. https://hdl.handle.net/21.15107/rcub_cherry_6205 .
Stefanović, Marija, Savić, Aleksa, Božić, Nataša, Vujčić, Zoran, Radosavljević, Jelena, "Electrophoretic assessment of recombinant λ- exonuclease production in different E. coli strains" in Zbornik apstrakata, VI Simpozijum Srpskog udruženja za proteomiku (SePA) “Razvoj i primena novih metoda proteomike”, 2. jun 2023. godine, Kragujevac (2023):23-23, https://hdl.handle.net/21.15107/rcub_cherry_6205 .