Recombinant production of native λ-exonuclease in different E. coli strains
Конференцијски прилог (Објављена верзија)
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Introduction: Lambda exonuclease (λ-exo), isolated from lambda bacteriophage, plays a crucial role in
DNA replication, repair and recombination. The enzyme specifically hydrolases double-stranded DNA
(dsDNA) in a highly processive manner in 5’�����3’ direction, yielding mononucleotides and single-stranded
DNA (ssDNA). This efficient unidirectional degradation makes it an invaluable tool in various molecular
biology techniques, including novel sequencing technologies. Hence, optimization of the expression
conditions is a prerequisite to achieving high-level production of λ-exo.
Methods: The N-terminally His-tagged λ-exo fusion construct with thrombin cleavage site (AddGene
#104531) was successfully transformed into five different E. coli strains (BL21(AI), Shuffle T7, C41(DE3),
C43(DE3), and BL21(DE3)). Expression was tested under three temperature regimes (20 ˚C, 30 ˚C, and 37
˚C) over time for enhanced soluble production. Crude extracts were analysed by SDS-PAGE for total pro...tein
expression, soluble and insoluble cytoplasmatic fractions. The exonuclease activity of the extracts
was monitored via in-house developed fluorescence-based screening assay. Optimal conditions for highyield
production were determined by densitometric analysis using NIH ImageJ software. The soluble and
active enzyme was produced on the large scale in a shaking flask culture under optimal conditions, and
purified to homogeneity from the soluble lysate via metal affinity chromatography.
Results:We identified E. coli BL21(AI), SHuffle T7, and C41(DE3) as good producers of recombinant λ-exo
and determined optimal conditions (30 ˚C, 6 h post-induction) for high-yield expression. The enzyme was
eluted from Ni2+-IDA-Sepharose 6B column in 300 mM imidazole and maintained its activity upon purification
assessed by an in-house developed fluorescence-based screening assay.
Conclusion: This study provides a scalable cost-effective approach for soluble λ-exo production in selected
E. coli strains. This expression system would be a helpful platform for development of high-yield
production of λ-exo, easing its exploitation in biotechnology and other scientific frontiers. Additionally,
we provided a valuable low-cost screening assay for monitoring exonuclease activity during each purification
step.
Кључне речи:
λ-exonuclease / recombinant technology / optimisation / E. coliИзвор:
CoMBoS2 – the Second Congress of Molecular Biologists of Serbia, Abstract Book – Trends in Molecular Biology, Special issue, 2023, 172-172Издавач:
- Institute of Molecular Genetics and Genetic Engineering (IMGGE), University of Belgrade
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/123456789/6208
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/123456789/6207 AB - Introduction: Lambda exonuclease (λ-exo), isolated from lambda bacteriophage, plays a crucial role in DNA replication, repair and recombination. The enzyme specifically hydrolases double-stranded DNA (dsDNA) in a highly processive manner in 5’�����3’ direction, yielding mononucleotides and single-stranded DNA (ssDNA). This efficient unidirectional degradation makes it an invaluable tool in various molecular biology techniques, including novel sequencing technologies. Hence, optimization of the expression conditions is a prerequisite to achieving high-level production of λ-exo. Methods: The N-terminally His-tagged λ-exo fusion construct with thrombin cleavage site (AddGene #104531) was successfully transformed into five different E. coli strains (BL21(AI), Shuffle T7, C41(DE3), C43(DE3), and BL21(DE3)). Expression was tested under three temperature regimes (20 ˚C, 30 ˚C, and 37 ˚C) over time for enhanced soluble production. Crude extracts were analysed by SDS-PAGE for total protein expression, soluble and insoluble cytoplasmatic fractions. The exonuclease activity of the extracts was monitored via in-house developed fluorescence-based screening assay. Optimal conditions for highyield production were determined by densitometric analysis using NIH ImageJ software. The soluble and active enzyme was produced on the large scale in a shaking flask culture under optimal conditions, and purified to homogeneity from the soluble lysate via metal affinity chromatography. Results:We identified E. coli BL21(AI), SHuffle T7, and C41(DE3) as good producers of recombinant λ-exo and determined optimal conditions (30 ˚C, 6 h post-induction) for high-yield expression. The enzyme was eluted from Ni2+-IDA-Sepharose 6B column in 300 mM imidazole and maintained its activity upon purification assessed by an in-house developed fluorescence-based screening assay. Conclusion: This study provides a scalable cost-effective approach for soluble λ-exo production in selected E. coli strains. This expression system would be a helpful platform for development of high-yield production of λ-exo, easing its exploitation in biotechnology and other scientific frontiers. Additionally, we provided a valuable low-cost screening assay for monitoring exonuclease activity during each purification step. PB - Institute of Molecular Genetics and Genetic Engineering (IMGGE), University of Belgrade C3 - CoMBoS2 – the Second Congress of Molecular Biologists of Serbia, Abstract Book – Trends in Molecular Biology, Special issue T1 - Recombinant production of native λ-exonuclease in different E. coli strains SP - 172 EP - 172 UR - https://hdl.handle.net/21.15107/rcub_cherry_6207 ER -
@conference{ author = "Stefanović, Marija and Savić, Aleksa and Božić, Nataša and Vujčić, Zoran and Radosavljević, Jelena", year = "2023", abstract = "Introduction: Lambda exonuclease (λ-exo), isolated from lambda bacteriophage, plays a crucial role in DNA replication, repair and recombination. The enzyme specifically hydrolases double-stranded DNA (dsDNA) in a highly processive manner in 5’�����3’ direction, yielding mononucleotides and single-stranded DNA (ssDNA). This efficient unidirectional degradation makes it an invaluable tool in various molecular biology techniques, including novel sequencing technologies. Hence, optimization of the expression conditions is a prerequisite to achieving high-level production of λ-exo. Methods: The N-terminally His-tagged λ-exo fusion construct with thrombin cleavage site (AddGene #104531) was successfully transformed into five different E. coli strains (BL21(AI), Shuffle T7, C41(DE3), C43(DE3), and BL21(DE3)). Expression was tested under three temperature regimes (20 ˚C, 30 ˚C, and 37 ˚C) over time for enhanced soluble production. Crude extracts were analysed by SDS-PAGE for total protein expression, soluble and insoluble cytoplasmatic fractions. The exonuclease activity of the extracts was monitored via in-house developed fluorescence-based screening assay. Optimal conditions for highyield production were determined by densitometric analysis using NIH ImageJ software. The soluble and active enzyme was produced on the large scale in a shaking flask culture under optimal conditions, and purified to homogeneity from the soluble lysate via metal affinity chromatography. Results:We identified E. coli BL21(AI), SHuffle T7, and C41(DE3) as good producers of recombinant λ-exo and determined optimal conditions (30 ˚C, 6 h post-induction) for high-yield expression. The enzyme was eluted from Ni2+-IDA-Sepharose 6B column in 300 mM imidazole and maintained its activity upon purification assessed by an in-house developed fluorescence-based screening assay. Conclusion: This study provides a scalable cost-effective approach for soluble λ-exo production in selected E. coli strains. This expression system would be a helpful platform for development of high-yield production of λ-exo, easing its exploitation in biotechnology and other scientific frontiers. Additionally, we provided a valuable low-cost screening assay for monitoring exonuclease activity during each purification step.", publisher = "Institute of Molecular Genetics and Genetic Engineering (IMGGE), University of Belgrade", journal = "CoMBoS2 – the Second Congress of Molecular Biologists of Serbia, Abstract Book – Trends in Molecular Biology, Special issue", title = "Recombinant production of native λ-exonuclease in different E. coli strains", pages = "172-172", url = "https://hdl.handle.net/21.15107/rcub_cherry_6207" }
Stefanović, M., Savić, A., Božić, N., Vujčić, Z.,& Radosavljević, J.. (2023). Recombinant production of native λ-exonuclease in different E. coli strains. in CoMBoS2 – the Second Congress of Molecular Biologists of Serbia, Abstract Book – Trends in Molecular Biology, Special issue Institute of Molecular Genetics and Genetic Engineering (IMGGE), University of Belgrade., 172-172. https://hdl.handle.net/21.15107/rcub_cherry_6207
Stefanović M, Savić A, Božić N, Vujčić Z, Radosavljević J. Recombinant production of native λ-exonuclease in different E. coli strains. in CoMBoS2 – the Second Congress of Molecular Biologists of Serbia, Abstract Book – Trends in Molecular Biology, Special issue. 2023;:172-172. https://hdl.handle.net/21.15107/rcub_cherry_6207 .
Stefanović, Marija, Savić, Aleksa, Božić, Nataša, Vujčić, Zoran, Radosavljević, Jelena, "Recombinant production of native λ-exonuclease in different E. coli strains" in CoMBoS2 – the Second Congress of Molecular Biologists of Serbia, Abstract Book – Trends in Molecular Biology, Special issue (2023):172-172, https://hdl.handle.net/21.15107/rcub_cherry_6207 .