TY  - 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/123456789/6206
AB  - Viral exonucleases play role in many processes essential for genome ma intenance,including DNA repair and recombination. Lambda exonuclease (λ -exo), isolated fromlambda bacteriophage, hydrolases double-stranded DNA (dsDNA) in the highly processivemanner 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 -moleculesequencing. Hence, optimization of the expression conditions is a prerequisite to achievehigh-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 conditionsfor efficient production of recombinant λ -exo. The N-terminally His -tagged λ-exo wassuccessfully expressed in E.coli BL21(AI), SHuffle T7, C41(DE3) and C43(DE3) strains inLB 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, aswell 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 satisfactoryyield. Our data suggest that densitometric analysis could serve as a powerful low-costscreening 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
UR  - https://hdl.handle.net/21.15107/rcub_cherry_6206
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 fromlambda bacteriophage, hydrolases double-stranded DNA (dsDNA) in the highly processivemanner 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 -moleculesequencing. Hence, optimization of the expression conditions is a prerequisite to achievehigh-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 conditionsfor efficient production of recombinant λ -exo. The N-terminally His -tagged λ-exo wassuccessfully expressed in E.coli BL21(AI), SHuffle T7, C41(DE3) and C43(DE3) strains inLB 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, aswell 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 satisfactoryyield. Our data suggest that densitometric analysis could serve as a powerful low-costscreening 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",
url = "https://hdl.handle.net/21.15107/rcub_cherry_6206"
}

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..
https://hdl.handle.net/21.15107/rcub_cherry_6206

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;.
https://hdl.handle.net/21.15107/rcub_cherry_6206 .

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),
https://hdl.handle.net/21.15107/rcub_cherry_6206 .

TY  - 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 .

TY  - 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 .

TY  - 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/123456789/6208
AB  - Introduction: Lambda exonuclease (λ-exo), isolated from lambda bacteriophage, plays a crucial role inDNA 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-strandedDNA (ssDNA). This efficient unidirectional degradation makes it an invaluable tool in various molecularbiology techniques, including novel sequencing technologies. Hence, optimization of the expressionconditions 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 proteinexpression, soluble and insoluble cytoplasmatic fractions. The exonuclease activity of the extractswas monitored via in-house developed fluorescence-based screening assay. Optimal conditions for highyieldproduction were determined by densitometric analysis using NIH ImageJ software. The soluble andactive enzyme was produced on the large scale in a shaking flask culture under optimal conditions, andpurified 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-exoand determined optimal conditions (30 ˚C, 6 h post-induction) for high-yield expression. The enzyme waseluted from Ni2+-IDA-Sepharose 6B column in 300 mM imidazole and maintained its activity upon purificationassessed by an in-house developed fluorescence-based screening assay.Conclusion: This study provides a scalable cost-effective approach for soluble λ-exo production in selectedE. coli strains. This expression system would be a helpful platform for development of high-yieldproduction 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 purificationstep.
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
UR  - https://hdl.handle.net/21.15107/rcub_cherry_6208
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 inDNA 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-strandedDNA (ssDNA). This efficient unidirectional degradation makes it an invaluable tool in various molecularbiology techniques, including novel sequencing technologies. Hence, optimization of the expressionconditions 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 proteinexpression, soluble and insoluble cytoplasmatic fractions. The exonuclease activity of the extractswas monitored via in-house developed fluorescence-based screening assay. Optimal conditions for highyieldproduction were determined by densitometric analysis using NIH ImageJ software. The soluble andactive enzyme was produced on the large scale in a shaking flask culture under optimal conditions, andpurified 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-exoand determined optimal conditions (30 ˚C, 6 h post-induction) for high-yield expression. The enzyme waseluted from Ni2+-IDA-Sepharose 6B column in 300 mM imidazole and maintained its activity upon purificationassessed by an in-house developed fluorescence-based screening assay.Conclusion: This study provides a scalable cost-effective approach for soluble λ-exo production in selectedE. coli strains. This expression system would be a helpful platform for development of high-yieldproduction 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 purificationstep.",
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",
url = "https://hdl.handle.net/21.15107/rcub_cherry_6208"
}

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..
https://hdl.handle.net/21.15107/rcub_cherry_6208

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;.
https://hdl.handle.net/21.15107/rcub_cherry_6208 .

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),
https://hdl.handle.net/21.15107/rcub_cherry_6208 .

TY  - THES
AU  - Stefanović, Marija
PY  - 2023
UR  - http://cherry.chem.bg.ac.rs/handle/123456789/6135
AB  - Lambda egzonukleaza (λ-exo), izolovana iz lambda bakteriofaga, igra kljuĉnu ulogu u replikaciji, 
popravci i rekombinaciji genoma λ- akteriofaga Ovaj enzim specifiĉno hidrolizuje dvolanĉanu 
DN dsDN na visoko procesivan naĉin u pravcu 5‟ 3‟, dajući pojedinaĉne mononukleotide i 
jednolanĉanu DN ssDN Sposobnost efikasne katalize unidirekcione hidrolize nukleinskih 
kiselina ĉini ga neprocenjivim alatom u razliĉitim metodama molekularne biotehnologije, 
ukljuĉujući nove tehnologije sekvenciranja kao što je sekvenciranje u nanoporama. Rekombinantna 
ekspresija enzima u bakteriji E. coli nudi moćnu platformu za efikasnu proizvodnju iotehnološki 
znaĉajnih enzima Optimizacija uslova ekspresije i preĉišćavanja je kljuĉni preduslov za postizanje 
visokog nivoa proizvodnje rekombinantnih proteina. Cilj ovog master rada bilo je uspostavljanje 
sistema za rekombinantnu proizvodnju λ-egzonukleaze u bakteriji E. coli na velikoj laboratorijskoj 
skali. Kao rezultat, sojevi E. coli BL21(AI), Shuffle T7 i C41(DE3) identifikovani kao dobri
produceri proteina od interesa i optimizovani su uslovi za solubilnu citoplazmatsku ekspresiju (30 
C, 6 h nakon indukovanja). Nativna rekom inantna egzonukleaza je uspešno proizvedena na velikoj 
laboratorijskoj skali (1 L kulture) u zadovoljavajućem prinosu, a onda i preĉišćena do maksimalno 
80% ĉistoće imobilizovanom metal afinitetnom hromatografijom. Dalja istraživanja i tre alo 
usmeriti ka prevazilaženju pro lema taloženja proteina nakon elucije sa kolone, što je dovodilo do 
nezanemarljivih gubitaka u prinosu.
T1  - Izazovi heterologe proizvodnje lambda egzonukleaze u bakteriji  Escherichia coli
SP  - 2
EP  - 73
UR  - https://hdl.handle.net/21.15107/rcub_cherry_6135
ER  - 

@mastersthesis{
author = "Stefanović, Marija",
year = "2023",
abstract = "Lambda egzonukleaza (λ-exo), izolovana iz lambda bakteriofaga, igra kljuĉnu ulogu u replikaciji, 
popravci i rekombinaciji genoma λ- akteriofaga Ovaj enzim specifiĉno hidrolizuje dvolanĉanu 
DN dsDN na visoko procesivan naĉin u pravcu 5‟ 3‟, dajući pojedinaĉne mononukleotide i 
jednolanĉanu DN ssDN Sposobnost efikasne katalize unidirekcione hidrolize nukleinskih 
kiselina ĉini ga neprocenjivim alatom u razliĉitim metodama molekularne biotehnologije, 
ukljuĉujući nove tehnologije sekvenciranja kao što je sekvenciranje u nanoporama. Rekombinantna 
ekspresija enzima u bakteriji E. coli nudi moćnu platformu za efikasnu proizvodnju iotehnološki 
znaĉajnih enzima Optimizacija uslova ekspresije i preĉišćavanja je kljuĉni preduslov za postizanje 
visokog nivoa proizvodnje rekombinantnih proteina. Cilj ovog master rada bilo je uspostavljanje 
sistema za rekombinantnu proizvodnju λ-egzonukleaze u bakteriji E. coli na velikoj laboratorijskoj 
skali. Kao rezultat, sojevi E. coli BL21(AI), Shuffle T7 i C41(DE3) identifikovani kao dobri
produceri proteina od interesa i optimizovani su uslovi za solubilnu citoplazmatsku ekspresiju (30 
C, 6 h nakon indukovanja). Nativna rekom inantna egzonukleaza je uspešno proizvedena na velikoj 
laboratorijskoj skali (1 L kulture) u zadovoljavajućem prinosu, a onda i preĉišćena do maksimalno 
80% ĉistoće imobilizovanom metal afinitetnom hromatografijom. Dalja istraživanja i tre alo 
usmeriti ka prevazilaženju pro lema taloženja proteina nakon elucije sa kolone, što je dovodilo do 
nezanemarljivih gubitaka u prinosu.",
title = "Izazovi heterologe proizvodnje lambda egzonukleaze u bakteriji  Escherichia coli",
pages = "2-73",
url = "https://hdl.handle.net/21.15107/rcub_cherry_6135"
}

Stefanović, M.. (2023). Izazovi heterologe proizvodnje lambda egzonukleaze u bakteriji  Escherichia coli. , 2-73.
https://hdl.handle.net/21.15107/rcub_cherry_6135

Stefanović M. Izazovi heterologe proizvodnje lambda egzonukleaze u bakteriji  Escherichia coli. 2023;:2-73.
https://hdl.handle.net/21.15107/rcub_cherry_6135 .

Stefanović, Marija, "Izazovi heterologe proizvodnje lambda egzonukleaze u bakteriji  Escherichia coli" (2023):2-73,
https://hdl.handle.net/21.15107/rcub_cherry_6135 .

TY  - CONF
AU  - Savić, Aleksa D.
AU  - Stefanović, Marija
AU  - Slović, Filip
AU  - Radosavljević, Jelena
PY  - 2023
UR  - http://cherry.chem.bg.ac.rs/handle/123456789/5972
AB  - PET hydrolases are enzymes that have been shown to act upon PET as a substrate. Theseenzymes usually adopt an α/β hydrolase fold and are from the classes of esterases, lipases,cutinases, and hydrolases.Here, we have done sequence alignment by ClustalW of the sequences corresponding tothe entries available in the PAZy database (pazy.eu) with the addition of a highly efficientI. sakaiensis PETase mutant W159H/S238F and analyzed the results. The aligned sequencesincluded several different well-aligned segments, which were as follows: 18 single-amino acidsegments, 13 two-amino acid segments, 10 three-amino-acid segments, 1 four-amino acidsegment, 1 six-amino acid segment and 1 eight-amino acid segment. Additionally, at position238, which is adjacent to a highly conserved His237, the most common amino acids wereF,T, S, Y, W, L and G, whereas at position 159, the most common amino acids were W, H, I andL, flanked by a conserved three- and eight-amino acid region. These positions seem to becritical for the improvement of the PET hydrolytic activity based on the comparison ofI. sakaiensis PETase mutant W159H/S238F and wt enzyme.Using AlphaFold 2.0 we have predicted the structures of all enzymes available in the databasewhose structures haven't been previously reported and the presence of the α/β hydrolasemotif has been observed. The sequences were also analyzed by SIAS (imed.med.ucm.es).
PB  - European federation of biotechnology
PB  - Asian federation of biotechnology
C3  - Biotechnology for a circular bioeconomy, 28-29 March 2023, AFOB-EFB Virtual Conference
T1  - Analysis of PET degrading enzymes by bioinfomatic tools
UR  - https://hdl.handle.net/21.15107/rcub_cherry_5972
ER  - 

@conference{
author = "Savić, Aleksa D. and Stefanović, Marija and Slović, Filip and Radosavljević, Jelena",
year = "2023",
abstract = "PET hydrolases are enzymes that have been shown to act upon PET as a substrate. Theseenzymes usually adopt an α/β hydrolase fold and are from the classes of esterases, lipases,cutinases, and hydrolases.Here, we have done sequence alignment by ClustalW of the sequences corresponding tothe entries available in the PAZy database (pazy.eu) with the addition of a highly efficientI. sakaiensis PETase mutant W159H/S238F and analyzed the results. The aligned sequencesincluded several different well-aligned segments, which were as follows: 18 single-amino acidsegments, 13 two-amino acid segments, 10 three-amino-acid segments, 1 four-amino acidsegment, 1 six-amino acid segment and 1 eight-amino acid segment. Additionally, at position238, which is adjacent to a highly conserved His237, the most common amino acids wereF,T, S, Y, W, L and G, whereas at position 159, the most common amino acids were W, H, I andL, flanked by a conserved three- and eight-amino acid region. These positions seem to becritical for the improvement of the PET hydrolytic activity based on the comparison ofI. sakaiensis PETase mutant W159H/S238F and wt enzyme.Using AlphaFold 2.0 we have predicted the structures of all enzymes available in the databasewhose structures haven't been previously reported and the presence of the α/β hydrolasemotif has been observed. The sequences were also analyzed by SIAS (imed.med.ucm.es).",
publisher = "European federation of biotechnology, Asian federation of biotechnology",
journal = "Biotechnology for a circular bioeconomy, 28-29 March 2023, AFOB-EFB Virtual Conference",
title = "Analysis of PET degrading enzymes by bioinfomatic tools",
url = "https://hdl.handle.net/21.15107/rcub_cherry_5972"
}

Savić, A. D., Stefanović, M., Slović, F.,& Radosavljević, J.. (2023). Analysis of PET degrading enzymes by bioinfomatic tools. in Biotechnology for a circular bioeconomy, 28-29 March 2023, AFOB-EFB Virtual Conference
European federation of biotechnology..
https://hdl.handle.net/21.15107/rcub_cherry_5972

Savić AD, Stefanović M, Slović F, Radosavljević J. Analysis of PET degrading enzymes by bioinfomatic tools. in Biotechnology for a circular bioeconomy, 28-29 March 2023, AFOB-EFB Virtual Conference. 2023;.
https://hdl.handle.net/21.15107/rcub_cherry_5972 .

Savić, Aleksa D., Stefanović, Marija, Slović, Filip, Radosavljević, Jelena, "Analysis of PET degrading enzymes by bioinfomatic tools" in Biotechnology for a circular bioeconomy, 28-29 March 2023, AFOB-EFB Virtual Conference (2023),
https://hdl.handle.net/21.15107/rcub_cherry_5972 .

TY  - CONF
AU  - Savić, Aleksa D.
AU  - Stefanović, Marija
AU  - Slović, Filip
AU  - Radosavljević, Jelena
PY  - 2023
UR  - http://cherry.chem.bg.ac.rs/handle/123456789/5971
AB  - PET hydrolases are enzymes that have been shown to act upon PET as a substrate. These
enzymes usually adopt an α/β hydrolase fold and are from the classes of esterases, lipases,
cutinases, and hydrolases.
Here, we have done sequence alignment by ClustalW of the sequences corresponding to
the entries available in the PAZy database (pazy.eu) with the addition of a highly efficient
I. sakaiensis PETase mutant W159H/S238F and analyzed the results. The aligned sequences
included several different well-aligned segments, which were as follows: 18 single-amino acid
segments, 13 two-amino acid segments, 10 three-amino-acid segments, 1 four-amino acid
segment, 1 six-amino acid segment and 1 eight-amino acid segment. Additionally, at position
238, which is adjacent to a highly conserved His237, the most common amino acids were
F,T, S, Y, W, L and G, whereas at position 159, the most common amino acids were W, H, I and
L, flanked by a conserved three- and eight-amino acid region. These positions seem to be
critical for the improvement of the PET hydrolytic activity based on the comparison of
I. sakaiensis PETase mutant W159H/S238F and wt enzyme.
Using AlphaFold 2.0 we have predicted the structures of all enzymes available in the database
whose structures haven't been previously reported and the presence of the α/β hydrolase
motif has been observed. The sequences were also analyzed by SIAS (imed.med.ucm.es).
PB  - European federation of biotechnology
PB  - Asian federation of biotechnology
C3  - Biotechnology for a circular bioeconomy, 28-29 March 2023, AFOB-EFB Virtual Conference
T1  - Analysis of PET degrading enzymes by bioinfomatic tools
SP  - 103
EP  - 103
UR  - https://hdl.handle.net/21.15107/rcub_cherry_5971
ER  - 

@conference{
author = "Savić, Aleksa D. and Stefanović, Marija and Slović, Filip and Radosavljević, Jelena",
year = "2023",
abstract = "PET hydrolases are enzymes that have been shown to act upon PET as a substrate. These
enzymes usually adopt an α/β hydrolase fold and are from the classes of esterases, lipases,
cutinases, and hydrolases.
Here, we have done sequence alignment by ClustalW of the sequences corresponding to
the entries available in the PAZy database (pazy.eu) with the addition of a highly efficient
I. sakaiensis PETase mutant W159H/S238F and analyzed the results. The aligned sequences
included several different well-aligned segments, which were as follows: 18 single-amino acid
segments, 13 two-amino acid segments, 10 three-amino-acid segments, 1 four-amino acid
segment, 1 six-amino acid segment and 1 eight-amino acid segment. Additionally, at position
238, which is adjacent to a highly conserved His237, the most common amino acids were
F,T, S, Y, W, L and G, whereas at position 159, the most common amino acids were W, H, I and
L, flanked by a conserved three- and eight-amino acid region. These positions seem to be
critical for the improvement of the PET hydrolytic activity based on the comparison of
I. sakaiensis PETase mutant W159H/S238F and wt enzyme.
Using AlphaFold 2.0 we have predicted the structures of all enzymes available in the database
whose structures haven't been previously reported and the presence of the α/β hydrolase
motif has been observed. The sequences were also analyzed by SIAS (imed.med.ucm.es).",
publisher = "European federation of biotechnology, Asian federation of biotechnology",
journal = "Biotechnology for a circular bioeconomy, 28-29 March 2023, AFOB-EFB Virtual Conference",
title = "Analysis of PET degrading enzymes by bioinfomatic tools",
pages = "103-103",
url = "https://hdl.handle.net/21.15107/rcub_cherry_5971"
}

Savić, A. D., Stefanović, M., Slović, F.,& Radosavljević, J.. (2023). Analysis of PET degrading enzymes by bioinfomatic tools. in Biotechnology for a circular bioeconomy, 28-29 March 2023, AFOB-EFB Virtual Conference
European federation of biotechnology., 103-103.
https://hdl.handle.net/21.15107/rcub_cherry_5971

Savić AD, Stefanović M, Slović F, Radosavljević J. Analysis of PET degrading enzymes by bioinfomatic tools. in Biotechnology for a circular bioeconomy, 28-29 March 2023, AFOB-EFB Virtual Conference. 2023;:103-103.
https://hdl.handle.net/21.15107/rcub_cherry_5971 .

Savić, Aleksa D., Stefanović, Marija, Slović, Filip, Radosavljević, Jelena, "Analysis of PET degrading enzymes by bioinfomatic tools" in Biotechnology for a circular bioeconomy, 28-29 March 2023, AFOB-EFB Virtual Conference (2023):103-103,
https://hdl.handle.net/21.15107/rcub_cherry_5971 .

TY  - THES
AU  - Stefanović, Marija D.
PY  - 2022
UR  - http://cherry.chem.bg.ac.rs/handle/123456789/5559
AB  - Nukleaze su strukturno i funkcionalno raznolika grupa enzima koja uključuje proteine i molekule RNA sa katalitičkom aktivnošću. Hidrolizuju fosfodiestarske veze nukleinskih kiselina sa 3’ ili 5’ kraja polinukleotidnog lanca. Sa biotehnološkog aspekta posebno značajne su termolabilne visoko procesivne egzonukleaze koje degraduju DNA do oligo- i mononukleotida. Ovi enzimi imaju primenu u pripremi uzoraka za kvantifikaciju hemijski i enzimski modfikovanim nukleotidima u genomu LC-MS/MS tehnikom. Cilj ovog rada bila je bioinformatička analiza fizičkohemijskih i funkcionalnih podataka o nespecifičnim nukleazama, kao i organizovanje izvučenih informacija u bazu podataka koja je kompatibilna za primenu u obuci modela u mašinskom učenju. Pored toga, cilj je bilo odrediti stepen homologije među izabranim predstavnicima enzima, kao i identifikovati evolutivno konzervirane strukturne motive. Dobijeni model bi se koristio za određivanje strukturnih specifičnosti koje doprinose visokoj procesivnosti egzonukleaza koje hidrolizuju nukleinske kiselina do mononukleotida. Kao rezulatat napravljena je baza 148 sekvencija sortiranih prema EC broju. Za svaki enzim određeni su fizičkohemijski parametri (teorijska pI, GRAVY, indeks nestabilnosti, molarna masa) i funkcionalni parametri (pH i temperaturni optimumi, kcat i Km) pomoću bioinformatičkih algoritama. Urađeno je višestruko poravnavanje grupa sekvencija koje sadrže konzervirane motive i među članovima je određen procenat sličnosti i identičnosti.
T1  - Bioinformatička analiza sekvencija nespecifičnih egzo- i endonukleaza
SP  - 1
EP  - 37
UR  - https://hdl.handle.net/21.15107/rcub_cherry_5559
ER  - 

@misc{
author = "Stefanović, Marija D.",
year = "2022",
abstract = "Nukleaze su strukturno i funkcionalno raznolika grupa enzima koja uključuje proteine i molekule RNA sa katalitičkom aktivnošću. Hidrolizuju fosfodiestarske veze nukleinskih kiselina sa 3’ ili 5’ kraja polinukleotidnog lanca. Sa biotehnološkog aspekta posebno značajne su termolabilne visoko procesivne egzonukleaze koje degraduju DNA do oligo- i mononukleotida. Ovi enzimi imaju primenu u pripremi uzoraka za kvantifikaciju hemijski i enzimski modfikovanim nukleotidima u genomu LC-MS/MS tehnikom. Cilj ovog rada bila je bioinformatička analiza fizičkohemijskih i funkcionalnih podataka o nespecifičnim nukleazama, kao i organizovanje izvučenih informacija u bazu podataka koja je kompatibilna za primenu u obuci modela u mašinskom učenju. Pored toga, cilj je bilo odrediti stepen homologije među izabranim predstavnicima enzima, kao i identifikovati evolutivno konzervirane strukturne motive. Dobijeni model bi se koristio za određivanje strukturnih specifičnosti koje doprinose visokoj procesivnosti egzonukleaza koje hidrolizuju nukleinske kiselina do mononukleotida. Kao rezulatat napravljena je baza 148 sekvencija sortiranih prema EC broju. Za svaki enzim određeni su fizičkohemijski parametri (teorijska pI, GRAVY, indeks nestabilnosti, molarna masa) i funkcionalni parametri (pH i temperaturni optimumi, kcat i Km) pomoću bioinformatičkih algoritama. Urađeno je višestruko poravnavanje grupa sekvencija koje sadrže konzervirane motive i među članovima je određen procenat sličnosti i identičnosti.",
title = "Bioinformatička analiza sekvencija nespecifičnih egzo- i endonukleaza",
pages = "1-37",
url = "https://hdl.handle.net/21.15107/rcub_cherry_5559"
}

Stefanović, M. D.. (2022). Bioinformatička analiza sekvencija nespecifičnih egzo- i endonukleaza. , 1-37.
https://hdl.handle.net/21.15107/rcub_cherry_5559

Stefanović MD. Bioinformatička analiza sekvencija nespecifičnih egzo- i endonukleaza. 2022;:1-37.
https://hdl.handle.net/21.15107/rcub_cherry_5559 .

Stefanović, Marija D., "Bioinformatička analiza sekvencija nespecifičnih egzo- i endonukleaza" (2022):1-37,
https://hdl.handle.net/21.15107/rcub_cherry_5559 .