TY  - CONF
AU  - Savić, Aleksa
AU  - Drulović, Nenad
AU  - Radosavljević, Jelena
PY  - 2023
UR  - http://cherry.chem.bg.ac.rs/handle/123456789/6179
AB  - Plastic materials have become indispensable in the modern world, with their extensive use resulting in their environmental accumulation. A promising solution for overcoming this ecological threat may be found in recombinantly produced plastic-degrading enzymes. Due to the complexity of the heterogeneous catalysis occuring during enzymatic PET hydrolysis, quantifying and comparing activities of such enzymes is rendered difficult. Here, we have assessed various assays documented in existing literature, employing different preparations of the purified recombinant Ideonella sakaiensis PETase mutant W159H/S238F (expressed from commercial plasmid Addgene #112203). The investigated methods were as follows: p-nitrophenyl acetate (pNA) hydrolysis assay, bis-(2-hydrohxyethyl)-terephthalate (BHET) agar and PET agar diffusion assays, and UV absorbance monitoring after PET particle and PET bottle cut-out hydrolysis. Additionally, we introduced an indirect colorimetric assay using the indicator pyrocatechol violet (PCV).
Our work reveals many advantages and problems for each of the tested methods. The pNA hydrolysis assay is the quickest, but many substances which are usually present in enzyme buffers and solutions tend to hydrolyse this compound (e.g. imidazole). It is also unspecific due to hydrolysis by other esterase enzymes. The BHET diffusion assay offers a great tool for activity comparison and estimation, with greater enzyme specificity. However, it is slow and accurate activity quantification is difficult. PET hydrolysis was conducted on in-house prepared PET particles with UV spectrophotometric measurement or by a diffusion assay. Due to the measuring wavelength (240 nm), the importance of proper blanking is critical, but accurate results can still be obtained. The sensitivity of the diffusion assay is much lower in comparison to the similar BHET assay. We also report on a modification of the phenol red indirect colorimetric assay using PCV as the indicator and PET particles as the substrate, which has not been previously described in existing literature.
PB  - Faculty of Chemistry, Serbian Biochemical Society
C3  - "Biochemistry in Biotechnology", Twelfth Conference, International scientific meeting, September 21-23, 2023, Belgrade, Serbia
T1  - Evaluation of an in-house developed colorimetric and other assays for PET-degrading activity
SP  - 105
EP  - 105
UR  - https://hdl.handle.net/21.15107/rcub_cherry_6179
ER  - 

@conference{
author = "Savić, Aleksa and Drulović, Nenad and Radosavljević, Jelena",
year = "2023",
abstract = "Plastic materials have become indispensable in the modern world, with their extensive use resulting in their environmental accumulation. A promising solution for overcoming this ecological threat may be found in recombinantly produced plastic-degrading enzymes. Due to the complexity of the heterogeneous catalysis occuring during enzymatic PET hydrolysis, quantifying and comparing activities of such enzymes is rendered difficult. Here, we have assessed various assays documented in existing literature, employing different preparations of the purified recombinant Ideonella sakaiensis PETase mutant W159H/S238F (expressed from commercial plasmid Addgene #112203). The investigated methods were as follows: p-nitrophenyl acetate (pNA) hydrolysis assay, bis-(2-hydrohxyethyl)-terephthalate (BHET) agar and PET agar diffusion assays, and UV absorbance monitoring after PET particle and PET bottle cut-out hydrolysis. Additionally, we introduced an indirect colorimetric assay using the indicator pyrocatechol violet (PCV).
Our work reveals many advantages and problems for each of the tested methods. The pNA hydrolysis assay is the quickest, but many substances which are usually present in enzyme buffers and solutions tend to hydrolyse this compound (e.g. imidazole). It is also unspecific due to hydrolysis by other esterase enzymes. The BHET diffusion assay offers a great tool for activity comparison and estimation, with greater enzyme specificity. However, it is slow and accurate activity quantification is difficult. PET hydrolysis was conducted on in-house prepared PET particles with UV spectrophotometric measurement or by a diffusion assay. Due to the measuring wavelength (240 nm), the importance of proper blanking is critical, but accurate results can still be obtained. The sensitivity of the diffusion assay is much lower in comparison to the similar BHET assay. We also report on a modification of the phenol red indirect colorimetric assay using PCV as the indicator and PET particles as the substrate, which has not been previously described in existing literature.",
publisher = "Faculty of Chemistry, Serbian Biochemical Society",
journal = ""Biochemistry in Biotechnology", Twelfth Conference, International scientific meeting, September 21-23, 2023, Belgrade, Serbia",
title = "Evaluation of an in-house developed colorimetric and other assays for PET-degrading activity",
pages = "105-105",
url = "https://hdl.handle.net/21.15107/rcub_cherry_6179"
}

Savić, A., Drulović, N.,& Radosavljević, J.. (2023). Evaluation of an in-house developed colorimetric and other assays for PET-degrading activity. in "Biochemistry in Biotechnology", Twelfth Conference, International scientific meeting, September 21-23, 2023, Belgrade, Serbia
Faculty of Chemistry, Serbian Biochemical Society., 105-105.
https://hdl.handle.net/21.15107/rcub_cherry_6179

Savić A, Drulović N, Radosavljević J. Evaluation of an in-house developed colorimetric and other assays for PET-degrading activity. in "Biochemistry in Biotechnology", Twelfth Conference, International scientific meeting, September 21-23, 2023, Belgrade, Serbia. 2023;:105-105.
https://hdl.handle.net/21.15107/rcub_cherry_6179 .

Savić, Aleksa, Drulović, Nenad, Radosavljević, Jelena, "Evaluation of an in-house developed colorimetric and other assays for PET-degrading activity" in "Biochemistry in Biotechnology", Twelfth Conference, International scientific meeting, September 21-23, 2023, Belgrade, Serbia (2023):105-105,
https://hdl.handle.net/21.15107/rcub_cherry_6179 .

TY  - CONF
AU  - Savić, Aleksa
AU  - Radosavljević, Jelena
PY  - 2023
UR  - http://cherry.chem.bg.ac.rs/handle/123456789/6180
UR  - http://cherry.chem.bg.ac.rs/handle/123456789/6225
AB  - The optimization of expression conditions is a tedious, but necessary procedure for thesuccessful efficient production of recombinant proteins. Design of experiment (DoE) savestime and resources by using statistics to carefully select only a few experimental points(expression conditions) to obtain information about the whole tested range of variedconditions, unlike the most commonly used one-factor-at-a-time (OFAT) method, whichrequires full imput space screening for optimization. Here, we have optimized theexpression conditions of His 6 and His8-mCerulean3-TEV-α-synuclein production from thethe pDUET vector, as well as the His 6-tagged protein from the pET-20b vector andexpressed into the periplasm. The proteins for which optimization was conducted wereexpressed in Escherichia coli BL21(DE3) and BL21(DE3)pLysS. We have used DoE toplan adequate experimental points for optimization according to a Box-Behnken design,with IPTG concentration, temperature and time variation. After collecting the cultures atgiven experimental points, they were analyzed by SDS-PAGE and densitometry, thenmodelled and statistically analysed. The importance of correct sample preparation and gelloads for densitometric analysis is evident according to the obtained results. The largestexpression level was obtained from the His 8-tagged protein coded by the pDUET vector inE. coli BL21(DE3). We have also used densitometry to analyze expression levels indifferent 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
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 thesuccessful efficient production of recombinant proteins. Design of experiment (DoE) savestime and resources by using statistics to carefully select only a few experimental points(expression conditions) to obtain information about the whole tested range of variedconditions, unlike the most commonly used one-factor-at-a-time (OFAT) method, whichrequires full imput space screening for optimization. Here, we have optimized theexpression conditions of His 6 and His8-mCerulean3-TEV-α-synuclein production from thethe pDUET vector, as well as the His 6-tagged protein from the pET-20b vector andexpressed into the periplasm. The proteins for which optimization was conducted wereexpressed in Escherichia coli BL21(DE3) and BL21(DE3)pLysS. We have used DoE toplan adequate experimental points for optimization according to a Box-Behnken design,with IPTG concentration, temperature and time variation. After collecting the cultures atgiven experimental points, they were analyzed by SDS-PAGE and densitometry, thenmodelled and statistically analysed. The importance of correct sample preparation and gelloads for densitometric analysis is evident according to the obtained results. The largestexpression level was obtained from the His 8-tagged protein coded by the pDUET vector inE. coli BL21(DE3). We have also used densitometry to analyze expression levels indifferent 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",
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..
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;.
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),
https://hdl.handle.net/21.15107/rcub_cherry_6180 .

TY  - 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 .

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  - 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  - CONF
AU  - Savić, Aleksa D.
AU  - Radosavljević, Jelena
PY  - 2023
UR  - http://cherry.chem.bg.ac.rs/handle/123456789/5973
AB  - Polyethylene terephthalate (PET) is a widely used plastic material. Due to its convenient physicochemical properties, it has become irreplaceable in many scientific, industrial, medical and everyday uses, leading to an accumulation of this material in the environment and initiating many ecological problems, especially in marine ecosystems. One of the solutions for overcoming this  ecological threat may be found in recombinantly produced PET degrading enzymes.
The genes encoding proteins with prominent PET hydrolyzing activity (PETases) that have been successfully produced in Escherichia coli are commercially available (Addgene #112203 and #162667). These genes encode Ideonella sakaiensis PETase mutant W159H/S238F, and the fusion of the wild-type enzyme to MHETase (I. sakaiensis mono-(2-hydroxyethyl)terephthalic acid hydrolyzing enzyme).
Initially, we have done sequence alignment by ClustalW of the sequences corresponding to the entries available in the PAZy database (pazy.eu/doku.php) that contains information on many PET-degrading enzymes. We have identified amino acid substitutions that might be of interest for mutation towards improving the PET hydrolytic activity of IsPETase: at position W159 substitutions into H, I and L and at position S238 substitutions into F, T, Y, W, L and G. Since we are aiming to produce all of the abovementioned (double) mutants, we used different bioinformatic tools to predict the expression solubility of the mutated enzymes. To evaluate the accuracy of the available tools we have tested the expression levels and solubility of IsPETase W159H/S238F and IsPETase-MHETase fusion in E. coli. The IsPETase W159H/S238F protein was expressed fully soluble only at 20 oC, whereas the larger (~92 kDa) IsPETase-MHETase fusion protein was insoluble in all tested conditions. NetSolP (services.healthtech.dtu.dk/services/NetSolP-1.0/) gave the most accurate solubility predictions for the tested proteins and we used it for prediction of the solubility of the aimed mutants.
PB  - University of Belgrade - Faculty of Geography
C3  - International Scientific Conference Green Agenda for Western Balkans, Belgrade
T1  - Solubility prediction of the PET hydrolyzing enzyme’s double mutants for production in Escherichia coli
SP  - 70
EP  - 70
UR  - https://hdl.handle.net/21.15107/rcub_cherry_5973
ER  - 

@conference{
author = "Savić, Aleksa D. and Radosavljević, Jelena",
year = "2023",
abstract = "Polyethylene terephthalate (PET) is a widely used plastic material. Due to its convenient physicochemical properties, it has become irreplaceable in many scientific, industrial, medical and everyday uses, leading to an accumulation of this material in the environment and initiating many ecological problems, especially in marine ecosystems. One of the solutions for overcoming this  ecological threat may be found in recombinantly produced PET degrading enzymes.
The genes encoding proteins with prominent PET hydrolyzing activity (PETases) that have been successfully produced in Escherichia coli are commercially available (Addgene #112203 and #162667). These genes encode Ideonella sakaiensis PETase mutant W159H/S238F, and the fusion of the wild-type enzyme to MHETase (I. sakaiensis mono-(2-hydroxyethyl)terephthalic acid hydrolyzing enzyme).
Initially, we have done sequence alignment by ClustalW of the sequences corresponding to the entries available in the PAZy database (pazy.eu/doku.php) that contains information on many PET-degrading enzymes. We have identified amino acid substitutions that might be of interest for mutation towards improving the PET hydrolytic activity of IsPETase: at position W159 substitutions into H, I and L and at position S238 substitutions into F, T, Y, W, L and G. Since we are aiming to produce all of the abovementioned (double) mutants, we used different bioinformatic tools to predict the expression solubility of the mutated enzymes. To evaluate the accuracy of the available tools we have tested the expression levels and solubility of IsPETase W159H/S238F and IsPETase-MHETase fusion in E. coli. The IsPETase W159H/S238F protein was expressed fully soluble only at 20 oC, whereas the larger (~92 kDa) IsPETase-MHETase fusion protein was insoluble in all tested conditions. NetSolP (services.healthtech.dtu.dk/services/NetSolP-1.0/) gave the most accurate solubility predictions for the tested proteins and we used it for prediction of the solubility of the aimed mutants.",
publisher = "University of Belgrade - Faculty of Geography",
journal = "International Scientific Conference Green Agenda for Western Balkans, Belgrade",
title = "Solubility prediction of the PET hydrolyzing enzyme’s double mutants for production in Escherichia coli",
pages = "70-70",
url = "https://hdl.handle.net/21.15107/rcub_cherry_5973"
}

Savić, A. D.,& Radosavljević, J.. (2023). Solubility prediction of the PET hydrolyzing enzyme’s double mutants for production in Escherichia coli. in International Scientific Conference Green Agenda for Western Balkans, Belgrade
University of Belgrade - Faculty of Geography., 70-70.
https://hdl.handle.net/21.15107/rcub_cherry_5973

Savić AD, Radosavljević J. Solubility prediction of the PET hydrolyzing enzyme’s double mutants for production in Escherichia coli. in International Scientific Conference Green Agenda for Western Balkans, Belgrade. 2023;:70-70.
https://hdl.handle.net/21.15107/rcub_cherry_5973 .

Savić, Aleksa D., Radosavljević, Jelena, "Solubility prediction of the PET hydrolyzing enzyme’s double mutants for production in Escherichia coli" in International Scientific Conference Green Agenda for Western Balkans, Belgrade (2023):70-70,
https://hdl.handle.net/21.15107/rcub_cherry_5973 .

TY  - CONF
AU  - Savić, Aleksa D.
AU  - Vidović, Marija
AU  - Radosavljević, Jelena
PY  - 2022
UR  - http://cherry.chem.bg.ac.rs/handle/123456789/5870
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, Belgrade, Serbia, 29th October, 2022. In: Book of Abstracts
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
SP  - 10
EP  - 10
UR  - https://hdl.handle.net/21.15107/rcub_cherry_5870
ER  - 

@conference{
author = "Savić, Aleksa D. 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, Belgrade, Serbia, 29th October, 2022. In: Book of Abstracts",
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_cherry_5870"
}

Savić, A. D., 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, Belgrade, Serbia, 29th October, 2022. In: Book of Abstracts
Serbian Chemical Society., 10-10.
https://hdl.handle.net/21.15107/rcub_cherry_5870

Savić AD, 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, Belgrade, Serbia, 29th October, 2022. In: Book of Abstracts. 2022;:10-10.
https://hdl.handle.net/21.15107/rcub_cherry_5870 .

Savić, Aleksa D., 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, Belgrade, Serbia, 29th October, 2022. In: Book of Abstracts (2022):10-10,
https://hdl.handle.net/21.15107/rcub_cherry_5870 .

TY  - CONF
AU  - Savić, Aleksa D.
AU  - Vidović, Marija
AU  - Radosavljević, Jelena
PY  - 2022
UR  - http://cherry.chem.bg.ac.rs/handle/123456789/5374
AB  - Fluorescentno obeleženi proteini su neprocenjivi alati u laboratorijskoj praksi za in vivo lokalizovanje i ispitivanje interakcija proteina. Dizajnirali smo vektor za ekspresiju mCerulean3 sa N-terminalnim heksahistidinskim obeleživacem fuzionisanim preko poliasparaginskog linkera i proteolitickog mesta za proteazu virusa graviranosti duvana (TEV) sa -sinukleinom. Ovaj konstrukt može se upotrebiti za proizvodnju -sinukleina nativne sekvence nakon proteolize TEV proteazom. Gen za mCerulean3 je nizom lancanih reakcija polimeraze (SOEing PCR) fuzionisan sa genom za -sinuklein i nakon amplifikacije ukloniran u plazmid pDUET-1. Escherichia coli BL21(DE3) je, nakon transformacije ovim konstruktom, upotrebljena za proizvodnju himernog proteina koji je zadržao fluorescentna svojstva sa prinosom od ~2 mg po litru medijuma nakon precišcavanja imobilizovanom metal-afinitetnom hromatografijom (elektroforetska cistoca: ~80%). Ovaj himerni protein je uspešno proteolizovan TEV proteazom.
AB  - Fluorescently labeled proteins are invaluable tools in laboratory practice to assess the in vivo localization and the interactions of proteins. Here we have designed an expression vector with an N-terminal hexahistidine-tagged mCerulean3 fused through a polyasparagine linker and the proteolytic site of tobacco etch virus protease (TEV) to - synuclein. This construct can be used to produce -synuclein of a native sequence after proteolysis with TEV protease. After fusion of the genes for mCerulean3 and -synuclein through a series of polymerase chain reactions (SOEing PCR), the resulting gene for the chimeric protein was cloned into the pDUET-1 plasmid. Escherichia coli BL21(DE3), upon transformation with this construct, can be used to produce the chimeric protein that retained the fluorescent properties of mCerulean3, with a yield of ~2 mg per liter of medium after purification by immobilized metal-affinity chromatography (electrophoretic purity: ~80%). This chimeric protein was successfully proteolyzed by TEV protease.
PB  - Beograd : Srpsko hemijsko društvo
C3  - 58. Savetovanje Srpskog hemijskog društva, Kratki izvodi radova, Beograd 9. i 10. jun 2022. godine
T1  - Kloniranje i ekspresija fluorescentno obeleženog )-sinukleina u bakteriji Escherichia coli
T1  - Cloning and expression of fluorescently labeled )-synuclein in Eschierichia coli
UR  - https://hdl.handle.net/21.15107/rcub_cherry_5374
ER  - 

@conference{
author = "Savić, Aleksa D. and Vidović, Marija and Radosavljević, Jelena",
year = "2022",
abstract = "Fluorescentno obeleženi proteini su neprocenjivi alati u laboratorijskoj praksi za in vivo lokalizovanje i ispitivanje interakcija proteina. Dizajnirali smo vektor za ekspresiju mCerulean3 sa N-terminalnim heksahistidinskim obeleživacem fuzionisanim preko poliasparaginskog linkera i proteolitickog mesta za proteazu virusa graviranosti duvana (TEV) sa -sinukleinom. Ovaj konstrukt može se upotrebiti za proizvodnju -sinukleina nativne sekvence nakon proteolize TEV proteazom. Gen za mCerulean3 je nizom lancanih reakcija polimeraze (SOEing PCR) fuzionisan sa genom za -sinuklein i nakon amplifikacije ukloniran u plazmid pDUET-1. Escherichia coli BL21(DE3) je, nakon transformacije ovim konstruktom, upotrebljena za proizvodnju himernog proteina koji je zadržao fluorescentna svojstva sa prinosom od ~2 mg po litru medijuma nakon precišcavanja imobilizovanom metal-afinitetnom hromatografijom (elektroforetska cistoca: ~80%). Ovaj himerni protein je uspešno proteolizovan TEV proteazom., Fluorescently labeled proteins are invaluable tools in laboratory practice to assess the in vivo localization and the interactions of proteins. Here we have designed an expression vector with an N-terminal hexahistidine-tagged mCerulean3 fused through a polyasparagine linker and the proteolytic site of tobacco etch virus protease (TEV) to - synuclein. This construct can be used to produce -synuclein of a native sequence after proteolysis with TEV protease. After fusion of the genes for mCerulean3 and -synuclein through a series of polymerase chain reactions (SOEing PCR), the resulting gene for the chimeric protein was cloned into the pDUET-1 plasmid. Escherichia coli BL21(DE3), upon transformation with this construct, can be used to produce the chimeric protein that retained the fluorescent properties of mCerulean3, with a yield of ~2 mg per liter of medium after purification by immobilized metal-affinity chromatography (electrophoretic purity: ~80%). This chimeric protein was successfully proteolyzed by TEV protease.",
publisher = "Beograd : Srpsko hemijsko društvo",
journal = "58. Savetovanje Srpskog hemijskog društva, Kratki izvodi radova, Beograd 9. i 10. jun 2022. godine",
title = "Kloniranje i ekspresija fluorescentno obeleženog )-sinukleina u bakteriji Escherichia coli, Cloning and expression of fluorescently labeled )-synuclein in Eschierichia coli",
url = "https://hdl.handle.net/21.15107/rcub_cherry_5374"
}

Savić, A. D., Vidović, M.,& Radosavljević, J.. (2022). Kloniranje i ekspresija fluorescentno obeleženog )-sinukleina u bakteriji Escherichia coli. in 58. Savetovanje Srpskog hemijskog društva, Kratki izvodi radova, Beograd 9. i 10. jun 2022. godine
Beograd : Srpsko hemijsko društvo..
https://hdl.handle.net/21.15107/rcub_cherry_5374

Savić AD, Vidović M, Radosavljević J. Kloniranje i ekspresija fluorescentno obeleženog )-sinukleina u bakteriji Escherichia coli. in 58. Savetovanje Srpskog hemijskog društva, Kratki izvodi radova, Beograd 9. i 10. jun 2022. godine. 2022;.
https://hdl.handle.net/21.15107/rcub_cherry_5374 .

Savić, Aleksa D., Vidović, Marija, Radosavljević, Jelena, "Kloniranje i ekspresija fluorescentno obeleženog )-sinukleina u bakteriji Escherichia coli" in 58. Savetovanje Srpskog hemijskog društva, Kratki izvodi radova, Beograd 9. i 10. jun 2022. godine (2022),
https://hdl.handle.net/21.15107/rcub_cherry_5374 .

TY  - CONF
AU  - Savić, Aleksa D.
AU  - Vidović, Marija
AU  - Radosavljević, Jelena
PY  - 2022
UR  - http://cherry.chem.bg.ac.rs/handle/123456789/5373
AB  - Fluorescentno obeleženi proteini su neprocenjivi alati u laboratorijskoj praksi za in vivo lokalizovanje i ispitivanje interakcija proteina. Dizajnirali smo vektor za ekspresiju mCerulean3 sa N-terminalnim heksahistidinskim obeleživacem fuzionisanim preko poliasparaginskog linkera i proteolitickog mesta za proteazu virusa graviranosti duvana (TEV) sa -sinukleinom. Ovaj konstrukt može se upotrebiti za proizvodnju -sinukleina nativne sekvence nakon proteolize TEV proteazom. Gen za mCerulean3 je nizom lancanih reakcija polimeraze (SOEing PCR) fuzionisan sa genom za -sinuklein i nakon amplifikacije ukloniran u plazmid pDUET-1. Escherichia coli BL21(DE3) je, nakon transformacije ovim konstruktom, upotrebljena za proizvodnju himernog proteina koji je zadržao fluorescentna svojstva sa prinosom od ~2 mg po litru medijuma nakon precišcavanja imobilizovanom metal-afinitetnom hromatografijom (elektroforetska cistoca: ~80%). Ovaj himerni protein je uspešno proteolizovan TEV proteazom.
AB  - Fluorescently labeled proteins are invaluable tools in laboratory practice to assess the in vivo localization and the interactions of proteins. Here we have designed an expression vector with an N-terminal hexahistidine-tagged mCerulean3 fused through a polyasparagine linker and the proteolytic site of tobacco etch virus protease (TEV) to - synuclein. This construct can be used to produce -synuclein of a native sequence after proteolysis with TEV protease. After fusion of the genes for mCerulean3 and -synuclein through a series of polymerase chain reactions (SOEing PCR), the resulting gene for the chimeric protein was cloned into the pDUET-1 plasmid. Escherichia coli BL21(DE3), upon transformation with this construct, can be used to produce the chimeric protein that retained the fluorescent properties of mCerulean3, with a yield of ~2 mg per liter of medium after purification by immobilized metal-affinity chromatography (electrophoretic purity: ~80%). This chimeric protein was successfully proteolyzed by TEV protease.
PB  - Beograd : Srpsko hemijsko društvo
C3  - 58. Savetovanje Srpskog hemijskog društva, Kratki izvodi radova, Beograd 9. i 10. jun 2022. godine
T1  - Kloniranje i ekspresija fluorescentno obeleženog )-sinukleina u bakteriji Escherichia coli
T1  - Cloning and expression of fluorescently labeled )-synuclein in Eschierichia coli
SP  - 68
EP  - 68
UR  - https://hdl.handle.net/21.15107/rcub_cherry_5373
ER  - 

@conference{
author = "Savić, Aleksa D. and Vidović, Marija and Radosavljević, Jelena",
year = "2022",
abstract = "Fluorescentno obeleženi proteini su neprocenjivi alati u laboratorijskoj praksi za in vivo lokalizovanje i ispitivanje interakcija proteina. Dizajnirali smo vektor za ekspresiju mCerulean3 sa N-terminalnim heksahistidinskim obeleživacem fuzionisanim preko poliasparaginskog linkera i proteolitickog mesta za proteazu virusa graviranosti duvana (TEV) sa -sinukleinom. Ovaj konstrukt može se upotrebiti za proizvodnju -sinukleina nativne sekvence nakon proteolize TEV proteazom. Gen za mCerulean3 je nizom lancanih reakcija polimeraze (SOEing PCR) fuzionisan sa genom za -sinuklein i nakon amplifikacije ukloniran u plazmid pDUET-1. Escherichia coli BL21(DE3) je, nakon transformacije ovim konstruktom, upotrebljena za proizvodnju himernog proteina koji je zadržao fluorescentna svojstva sa prinosom od ~2 mg po litru medijuma nakon precišcavanja imobilizovanom metal-afinitetnom hromatografijom (elektroforetska cistoca: ~80%). Ovaj himerni protein je uspešno proteolizovan TEV proteazom., Fluorescently labeled proteins are invaluable tools in laboratory practice to assess the in vivo localization and the interactions of proteins. Here we have designed an expression vector with an N-terminal hexahistidine-tagged mCerulean3 fused through a polyasparagine linker and the proteolytic site of tobacco etch virus protease (TEV) to - synuclein. This construct can be used to produce -synuclein of a native sequence after proteolysis with TEV protease. After fusion of the genes for mCerulean3 and -synuclein through a series of polymerase chain reactions (SOEing PCR), the resulting gene for the chimeric protein was cloned into the pDUET-1 plasmid. Escherichia coli BL21(DE3), upon transformation with this construct, can be used to produce the chimeric protein that retained the fluorescent properties of mCerulean3, with a yield of ~2 mg per liter of medium after purification by immobilized metal-affinity chromatography (electrophoretic purity: ~80%). This chimeric protein was successfully proteolyzed by TEV protease.",
publisher = "Beograd : Srpsko hemijsko društvo",
journal = "58. Savetovanje Srpskog hemijskog društva, Kratki izvodi radova, Beograd 9. i 10. jun 2022. godine",
title = "Kloniranje i ekspresija fluorescentno obeleženog )-sinukleina u bakteriji Escherichia coli, Cloning and expression of fluorescently labeled )-synuclein in Eschierichia coli",
pages = "68-68",
url = "https://hdl.handle.net/21.15107/rcub_cherry_5373"
}

Savić, A. D., Vidović, M.,& Radosavljević, J.. (2022). Kloniranje i ekspresija fluorescentno obeleženog )-sinukleina u bakteriji Escherichia coli. in 58. Savetovanje Srpskog hemijskog društva, Kratki izvodi radova, Beograd 9. i 10. jun 2022. godine
Beograd : Srpsko hemijsko društvo., 68-68.
https://hdl.handle.net/21.15107/rcub_cherry_5373

Savić AD, Vidović M, Radosavljević J. Kloniranje i ekspresija fluorescentno obeleženog )-sinukleina u bakteriji Escherichia coli. in 58. Savetovanje Srpskog hemijskog društva, Kratki izvodi radova, Beograd 9. i 10. jun 2022. godine. 2022;:68-68.
https://hdl.handle.net/21.15107/rcub_cherry_5373 .

Savić, Aleksa D., Vidović, Marija, Radosavljević, Jelena, "Kloniranje i ekspresija fluorescentno obeleženog )-sinukleina u bakteriji Escherichia coli" in 58. Savetovanje Srpskog hemijskog društva, Kratki izvodi radova, Beograd 9. i 10. jun 2022. godine (2022):68-68,
https://hdl.handle.net/21.15107/rcub_cherry_5373 .

TY  - THES
AU  - Savić, Aleksa D.
PY  - 2022
UR  - http://cherry.chem.bg.ac.rs/handle/123456789/5566
AB  - Alfa-sinuklein je protein zastupljen u nervnom tkivu, dominantno u završecima presinaptičih neurona. Agregati ovog proteina pronađeni su u nervnom tkivu brojnih pacijenata sa bolestima koje se pod jednim imenom označavaju α-sinukleinopatijama, a od kojih je najznačajnija Parkinsonova bolest. Formiranje ovih agregata smatra se usko povezanim sa etiologijom ove bolesti i zbog toga je značajno proizvesti i prečistiti ovaj protein u velikim količinama u cilju in vitro, a potencijalno i in vivo istraživanja. Koristeći komercijalni vektor pDUET-1-α-synuclein-mCerulean3-His6, u našoj laboratoriji su prethodnim radom proizvedena tri vektora za ekspresiju α-sinukleina u bakteriji Escherichia coli. U ovom radu je opisana optimizacija uslova ekspresije fluorescentno obeleženog humanog α-sinukleina sa sva tri plazmida transformisanih u sojeve E. coli BL21(DE3), odnosno BL21(DE3)pLysS, a koristeći metodologiju odzivne površine (RSM). Kombinacija plazmida i soja koja je pokazala najveći nivo ekspresije upotrebljena je za ekspresiju proteina od interesa na velikoj skali. Takođe, pokazano je da se dobijeni himerni protein nakon prečišćavanja imobilizovanom metal-afinitetnom hromatografijom može specifično hidrolizovati TEV proteazom. Nakon toga, α-sinuklein nativne sekvence dobijen proteolizom je grubo prečisćen ponovljenom metal-afinitetnom hromatografijom. Pokazano je i da prilikom zagrevanja himernog proteina ne dolazi do njegove značajne denaturacije i taloženja, što se može upotrebiti za postizanje značajnog prečišćenja jednim dodatnim korakom.
T1  - Optimizacija uslova ekspresije fluorescentno obeleženog humanog α-sinukleina u bakteriji Escherichia coli
SP  - 1
EP  - 84
UR  - https://hdl.handle.net/21.15107/rcub_cherry_5566
ER  - 

@mastersthesis{
author = "Savić, Aleksa D.",
year = "2022",
abstract = "Alfa-sinuklein je protein zastupljen u nervnom tkivu, dominantno u završecima presinaptičih neurona. Agregati ovog proteina pronađeni su u nervnom tkivu brojnih pacijenata sa bolestima koje se pod jednim imenom označavaju α-sinukleinopatijama, a od kojih je najznačajnija Parkinsonova bolest. Formiranje ovih agregata smatra se usko povezanim sa etiologijom ove bolesti i zbog toga je značajno proizvesti i prečistiti ovaj protein u velikim količinama u cilju in vitro, a potencijalno i in vivo istraživanja. Koristeći komercijalni vektor pDUET-1-α-synuclein-mCerulean3-His6, u našoj laboratoriji su prethodnim radom proizvedena tri vektora za ekspresiju α-sinukleina u bakteriji Escherichia coli. U ovom radu je opisana optimizacija uslova ekspresije fluorescentno obeleženog humanog α-sinukleina sa sva tri plazmida transformisanih u sojeve E. coli BL21(DE3), odnosno BL21(DE3)pLysS, a koristeći metodologiju odzivne površine (RSM). Kombinacija plazmida i soja koja je pokazala najveći nivo ekspresije upotrebljena je za ekspresiju proteina od interesa na velikoj skali. Takođe, pokazano je da se dobijeni himerni protein nakon prečišćavanja imobilizovanom metal-afinitetnom hromatografijom može specifično hidrolizovati TEV proteazom. Nakon toga, α-sinuklein nativne sekvence dobijen proteolizom je grubo prečisćen ponovljenom metal-afinitetnom hromatografijom. Pokazano je i da prilikom zagrevanja himernog proteina ne dolazi do njegove značajne denaturacije i taloženja, što se može upotrebiti za postizanje značajnog prečišćenja jednim dodatnim korakom.",
title = "Optimizacija uslova ekspresije fluorescentno obeleženog humanog α-sinukleina u bakteriji Escherichia coli",
pages = "1-84",
url = "https://hdl.handle.net/21.15107/rcub_cherry_5566"
}

Savić, A. D.. (2022). Optimizacija uslova ekspresije fluorescentno obeleženog humanog α-sinukleina u bakteriji Escherichia coli. , 1-84.
https://hdl.handle.net/21.15107/rcub_cherry_5566

Savić AD. Optimizacija uslova ekspresije fluorescentno obeleženog humanog α-sinukleina u bakteriji Escherichia coli. 2022;:1-84.
https://hdl.handle.net/21.15107/rcub_cherry_5566 .

Savić, Aleksa D., "Optimizacija uslova ekspresije fluorescentno obeleženog humanog α-sinukleina u bakteriji Escherichia coli" (2022):1-84,
https://hdl.handle.net/21.15107/rcub_cherry_5566 .

TY  - THES
AU  - Savić, Aleksa D.
PY  - 2021
UR  - https://cherry.chem.bg.ac.rs/handle/123456789/4736
AB  - Humani α-sinuklein je protein zastupljen u završecima presinaptičkih neurona. Agregati ovog proteina su pronađeni u okviru amiloidnih fibrila i Lewy-jevih tela u mozgovima pacijenata sa Alchajmerovom, Parkinsonovom i drugim bolestima i smatra se da su usko povezani sa etiologijom ovih bolesti. Da bi se ispitale priroda ovog proteina i njegove oligomerizacije in vitro potrebne su velike količine prečišćenog proteina. U ovom radu opisana je konstrukcija vektora za ekspresiju humanog  α-sinukleina fluorescentno obeleženog mCerulean3 proteinom sa His6, odnosno His8 tagom i uvedenim proteolitičkim mestom za TEV proteazu, koji nakon digestije oslobađa  α-sinuklein sa nativom N-terminalnom aminokiselinom, a polazeći od komercijalno dostupnog pDUET-A- α-sinuclein-mCerulean3 vektora. Za dobijanje ekspresionog konstrukta, prvo je sintetisan insert PCR-om iz više ciklusa, nakon čega su PCR proizvodi subklonirani u pET-20b vektor, a zatim prebačeni u pDUET vektor. Sekvenca inserta u dobijenim konstruktima je potvrđena sekvenciranjem sa opdgovarajućim prajmerima.
T1  - Kloniranje fluorescentno obeleženog humanog α-sinukleina u vektore za ekspresiju u bakteriji Escherichia coli
SP  - 1
EP  - 69
UR  - https://hdl.handle.net/21.15107/rcub_cherry_4736
ER  - 

@misc{
author = "Savić, Aleksa D.",
year = "2021",
abstract = "Humani α-sinuklein je protein zastupljen u završecima presinaptičkih neurona. Agregati ovog proteina su pronađeni u okviru amiloidnih fibrila i Lewy-jevih tela u mozgovima pacijenata sa Alchajmerovom, Parkinsonovom i drugim bolestima i smatra se da su usko povezani sa etiologijom ovih bolesti. Da bi se ispitale priroda ovog proteina i njegove oligomerizacije in vitro potrebne su velike količine prečišćenog proteina. U ovom radu opisana je konstrukcija vektora za ekspresiju humanog  α-sinukleina fluorescentno obeleženog mCerulean3 proteinom sa His6, odnosno His8 tagom i uvedenim proteolitičkim mestom za TEV proteazu, koji nakon digestije oslobađa  α-sinuklein sa nativom N-terminalnom aminokiselinom, a polazeći od komercijalno dostupnog pDUET-A- α-sinuclein-mCerulean3 vektora. Za dobijanje ekspresionog konstrukta, prvo je sintetisan insert PCR-om iz više ciklusa, nakon čega su PCR proizvodi subklonirani u pET-20b vektor, a zatim prebačeni u pDUET vektor. Sekvenca inserta u dobijenim konstruktima je potvrđena sekvenciranjem sa opdgovarajućim prajmerima.",
title = "Kloniranje fluorescentno obeleženog humanog α-sinukleina u vektore za ekspresiju u bakteriji Escherichia coli",
pages = "1-69",
url = "https://hdl.handle.net/21.15107/rcub_cherry_4736"
}

Savić, A. D.. (2021). Kloniranje fluorescentno obeleženog humanog α-sinukleina u vektore za ekspresiju u bakteriji Escherichia coli. , 1-69.
https://hdl.handle.net/21.15107/rcub_cherry_4736

Savić AD. Kloniranje fluorescentno obeleženog humanog α-sinukleina u vektore za ekspresiju u bakteriji Escherichia coli. 2021;:1-69.
https://hdl.handle.net/21.15107/rcub_cherry_4736 .

Savić, Aleksa D., "Kloniranje fluorescentno obeleženog humanog α-sinukleina u vektore za ekspresiju u bakteriji Escherichia coli" (2021):1-69,
https://hdl.handle.net/21.15107/rcub_cherry_4736 .