Purification of recombinant GFP produced by Agrobacterum-mediated transient expression in Nicotiana excelsior
Green fluorescent protein (GFP) is commonly used as a reporter protein in a wide range of biological experiments. The efficient protocol of Agrobacterium mediated transient expression in Nicotiana excelsior was applied for quick preparative production of recombinant GFP. The protein purification sch...
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nasplib_isofts_kiev_ua-123456789-80792025-02-09T15:59:51Z Purification of recombinant GFP produced by Agrobacterum-mediated transient expression in Nicotiana excelsior Sindarovska, Y.R. Sheludko, Y.V. Gerasymenko, I.M. Bannikova, M.A. Kuchuk, N.V. Оригинальные работы Green fluorescent protein (GFP) is commonly used as a reporter protein in a wide range of biological experiments. The efficient protocol of Agrobacterium mediated transient expression in Nicotiana excelsior was applied for quick preparative production of recombinant GFP. The protein purification scheme has been developed and included ammonium sulfate precipitation and Q sepharose anion exchange chromatography. It results in obtaining of a fraction with about 85 % GFP homogeneity and the protein yield of about 75 %. Зелений флуоресцентний білок (GFP) часто використовують як репортерний білок у різних галузях біологічних досліджень. Ефективний протокол Agrobacterium-опосередкованої транзієнтної експресії в Nicotiana excelsior було використано для швидкого одержання препаративної кількості рекомбінантного GFP. Розроблено схему очищення рекомбінантного білка, яка включає стадію осадження сульфатом амонію та іонообмінну хроматографію на сорбенті Q-sepharose. В результаті очищення було отримано фракцію білків з вмістом GFP близько 85 %. В ході очищення було отримано близько 75 % від початкової кількості рекомбінантного GFP. Зеленый флюоресцентный белок (GFP) часто используется в качестве маркерного белка в разных областях биологических исследований. Эффективный протокол Agrobacterium-опосредованной транзиентной экспрессии в Nicotiana excelsior был использован для быстрого получения препаративных количеств рекомбинантного GFP. Разработана схема очистки рекомбинантного белка, включающая стадию осаждения сульфатом аммония и ионообменную хроматографию на сорбенте Q-sepharose. В результате очистки была получена фракция белков с содержанием GFP около 85 %. В ходе очистки было выделено около 75 % от начального количества рекомбинантного GFP. The authors are grateful to the Ministry for Education and Science of Ukraine and National Academy of Science (grant DNTP 3.2. DP/148–2003 and 15K-2004) for financial support. 2008 Article Purification of recombinant GFP produced by Agrobacterum-mediated transient expression in Nicotiana excelsior / Y.R. Sindarovska, Y.V. Sheludko, I.M. Gerasymenko, M.A. Bannikova, N.V. Kuchuk // Цитология и генетика. — 2008. — Т. 42, № 2. — С. 16-20. — Бібліогр.: 20 назв. — англ. 0564-3783 https://nasplib.isofts.kiev.ua/handle/123456789/8079 57.085.2 + 582.926.2 + 577.21 en application/pdf Інститут клітинної біології та генетичної інженерії НАН України |
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Оригинальные работы Оригинальные работы |
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Оригинальные работы Оригинальные работы Sindarovska, Y.R. Sheludko, Y.V. Gerasymenko, I.M. Bannikova, M.A. Kuchuk, N.V. Purification of recombinant GFP produced by Agrobacterum-mediated transient expression in Nicotiana excelsior |
| description |
Green fluorescent protein (GFP) is commonly used as a reporter protein in a wide range of biological experiments. The efficient protocol of Agrobacterium mediated transient expression in Nicotiana excelsior was applied for quick preparative production of recombinant GFP. The protein purification scheme has been developed and included ammonium sulfate precipitation and Q sepharose anion exchange chromatography. It results in obtaining of a fraction with about 85 % GFP homogeneity and the protein yield of about 75 %. |
| format |
Article |
| author |
Sindarovska, Y.R. Sheludko, Y.V. Gerasymenko, I.M. Bannikova, M.A. Kuchuk, N.V. |
| author_facet |
Sindarovska, Y.R. Sheludko, Y.V. Gerasymenko, I.M. Bannikova, M.A. Kuchuk, N.V. |
| author_sort |
Sindarovska, Y.R. |
| title |
Purification of recombinant GFP produced by Agrobacterum-mediated transient expression in Nicotiana excelsior |
| title_short |
Purification of recombinant GFP produced by Agrobacterum-mediated transient expression in Nicotiana excelsior |
| title_full |
Purification of recombinant GFP produced by Agrobacterum-mediated transient expression in Nicotiana excelsior |
| title_fullStr |
Purification of recombinant GFP produced by Agrobacterum-mediated transient expression in Nicotiana excelsior |
| title_full_unstemmed |
Purification of recombinant GFP produced by Agrobacterum-mediated transient expression in Nicotiana excelsior |
| title_sort |
purification of recombinant gfp produced by agrobacterum-mediated transient expression in nicotiana excelsior |
| publisher |
Інститут клітинної біології та генетичної інженерії НАН України |
| publishDate |
2008 |
| topic_facet |
Оригинальные работы |
| url |
https://nasplib.isofts.kiev.ua/handle/123456789/8079 |
| citation_txt |
Purification of recombinant GFP produced by Agrobacterum-mediated transient expression in Nicotiana excelsior / Y.R. Sindarovska, Y.V. Sheludko, I.M. Gerasymenko, M.A. Bannikova, N.V. Kuchuk // Цитология и генетика. — 2008. — Т. 42, № 2. — С. 16-20. — Бібліогр.: 20 назв. — англ. |
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| first_indexed |
2025-11-27T19:44:53Z |
| last_indexed |
2025-11-27T19:44:53Z |
| _version_ |
1849974016496893952 |
| fulltext |
ISSN 0564–3783. Цитология и генетика. 2008. № 216
Green fluorescent protein (GFP) is commonly used as a
reporter protein in a wide range of biological experiments. The
efficient protocol of Agrobacterium�mediated transient expres�
sion in Nicotiana excelsior was applied for quick preparative
production of recombinant GFP. The protein purification
scheme has been developed and included ammonium sulfate
precipitation and Q�sepharose anion�exchange chromatogra�
phy. It results in obtaining of a fraction with about 85 % GFP
homogeneity and the protein yield of about 75 %.
Introduction. During the recent decade green
fluorescent protein (GFP) has become one of the
most popular in vivo marker molecule used in great
variety of biological and medical experiments, e.g.
researches on recombinant protein expression in
different cell systems, studies on promoter activi�
ties, protein targeting, localization, kinetics and
functional analysis of cytoskeleton and cytoskele�
ton�associated proteins etc [1, 2]. At the present
time GFP gene can be regarded as a substitution of
β�glucuronidase gene, which is widely used as a
reporter (gene) in transformed plants. In contrast
to GUS gene product, GFP can be directly moni�
tored or quantified in living cells without destruc�
tive tests.
GFP is derived from jellyfish Aequorea victoria
and contains a chromophore which does not
require any substrates or cofactors for fluorescence.
After cloning GFP has undergone substantial mod�
ifications that resulted in high expression rate,
increased fluorescence, stability and low toxicity
for a wide range of hosts, including plant cells
[3–5]. The present GFP�based protocols include
qualitative and quantitative analyses of GFP
expression by detection of its fluorescence from the
cellular level to the whole plant level [1, 2]. For cor�
rect quantitation considerable amount of purified
GFP is necessary for building of calibration curve.
Transient expression of foreign genes is a
recently developed method allowing production of
large amount of recombinant proteins within a very
short (days) time [6]. It occurs without stable inte�
gration of foreign DNA into the host genome. Our
recent work describes optimization of protocol for
rapid and high�scale production of recombinant
GFP using transient expression in Nicotiana
species [7].
Although numerous publications exist con�
cerning recombinant GFP production and purifi�
cation, most of them describe purification of GFP
from bacterial source. It includes tag�based proto�
cols [8, 9], which not always allow to separate
improperly folded or cyclized GFP from correctly
folded form of this protein [10], as well as size
exclusion chromatography [11], chromatofocus�
ing [12], organic extraction [11] etc. On the other
hand, GFP production and purification from plant
source under physiological conditions using ion�
exchange chromatography may be regarded as an
alternative method (including GFP purification
from constitutively transformed plant tissue). In
the present work we describe a scheme including
УДК 57.085.2 + 582.926.2 + 577.21
Y.R. SINDAROVSKA, Y.V. SHELUDKO,
I.M. GERASYMENKO, M.A. BANNIKOVA, N.V. KUCHUK
Institute of Cell Biology and Genetic Engineering,
Zabolotnogo str. 148, Kyiv 03680, Ukraine
E�mail: ysheludko@ukr.net
PURIFICATION OF RECOMBINANT
GFP PRODUCED
BY AGROBACTERUM.MEDIATED
TRANSIENT EXPRESSION
IN NICOTIANA EXCELSIOR
© Y.R. SINDAROVSKA, Y.V. SHELUDKO, I.M. GERASYMENKO,
M.A. BANNIKOVA, N.V. KUCHUK, 2008
ІSSN 0564–3783. Цитология и генетика. 2008. № 2 17
Purification of recombinant GFP produced by Agrobacterium(mediated
two stages of protein purification: ammonium sul�
fate precipitation and Q�sepharose anion�exchange
chromatography. It results in obtaining of fraction
with about 85 % GFP purity and the yield of the
initial fluorescent protein of about 75 %.
Materials and methods. Plant materials. Seeds of
Nicotiana excelsior J.M. Black were obtained from
the National Germplasm Bank of World Flora of the
Institute of Cell Biology and Genetic Engineering
(Kyiv, Ukraine). Plants were grown in greenhouse
at 20–25 °С and 14 h light period (3000–4000 lux).
Bacterial strains and genetic constructs. Con�
structs pICH10881, pICH10570 and pICH7410
represent a viral�based module system carrying
GFP gene which is described in details in the
recent publications [7, 13, 14]. The plasmid
pICH6692 [15] contained the gene of the p19 pro�
tein of tomato bushy stunt virus, a suppressor of
post�transcriptional gene silencing [16]. All the
mentioned plasmids were generously donated by
Icon Genetics GmbH (Halle/Saale, Germany).
Agrobacterium tumefaciens strain GV3101 trans�
formed with individual constructs was grown
overnight in LB medium supplemented with 50
mg/L rifampicin and 50 mg/L carbenicilin or
kanamicyn, and 100 µM acetosyringone.
Transient expression assay. Plant infiltration was
performed as it was described by Schob et al. [17]
with several modifications [7, 13]: A. tumefaciens
cells of overnight culture were centrifuged (5 min,
5000 g) and resuspended in the infiltration buffer
(10 mM MES, pH 5.5; 10 mM MgSO4; 100 µM
acetosyringone). The Agrobacterium suspensions
harboring different plasmid vectors were mixed in
the equal volumes so that the final optical density
(OD 600) of each suspension in the infiltration
buffer amounted 0.5 for viral�based cassette
(pICH10881, pICH10570, pICH7410) coexpressed
with the suppressor of silencing (pICH6692). For
typical assay the leaves of greenhouse grown N.
excelsior plants were filled with Agrobacterium mix�
ture by using a syringe without a needle. After
infiltration, the plants were further grown under
greenhouse conditions.
Protein extraction and precipitation. Accumu�
lation of GFP in the infiltrated leaves was moni�
tored at the 18th day after infiltration with a hand�
held black ray lamp (UVP, Upland, USA) and the
fluorescent areas were cut out. For typical assay
leaf tissue (app. 15 g) was grounded in blender for
1–2 min with 75 mL of buffer A (100 mM KPi,
pH 7.8; 5 mM EDTA; 10 mM β�mercaptoethanol).
After filtration through 3 layers of nylon, extract
was centrifuged at 10000 g for 15 min at +4 °С.
The supernatant was collected and used for
ammonium sulfate precipitation.
For protein precipitation, ammonium sulfate
was added to the clarified extract (19.47 g
(NH4)2SO4 per 66 mL of solution) up to 50 % of
saturation and incubated in ice for 1 h. After cen�
trifugation (15 min, 10000 g, +4 °С) ammonium
sulfate was added to the supernatant (9.4 g
(NH4)2SO4 per 74 mL of solution) to a final con�
centration of 70 % saturation. After 1 h incubation
the solution was centrifuged again, and the precip�
itated proteins were dissolved in 5 mL of buffer B
(20 mM KPi, pH 7.8; 2 mM EDTA; 10 mM β�
mercaptoethanol). The protein extract was dia�
lyzed two times against 2 L of buffer B.
Q�Sepharose chromatography. The dialyzed pro�
teins were filtered through 0.44 µm membrane and
applied onto a column filled with Q�Sepharose FF
(Amersham Biosciences, Piscataway, USA; volume
11 mL) equilibrated with buffer B. After washing
the column with 4 column volumes of buffer B, the
proteins were eluted with a linear NaCl gradient
(18 column volumes, 0–1 M NaCl) prepared from
buffer B and C (20 mM KPi, pH 7.8; 2 mM
EDTA; 10 mM β�mercaptoethanol, 1 M NaCl) at
a flow rate of 3 mL/min collecting 5 mL fractions.
GFP analysis. GFP content was calculated by
measurements of fluorescence intensity in dilu�
tions of protein extracts using fluorescence spec�
trophotometer Hitachi 850 (Hitachi, Japan) (exci�
tation at 395 nm, emission at 509 nm) on the basis
of standard values (GFP standard was generously
granted by Icon Genetics GmbH (Halle/Saale,
Germany)). The background fluorescence of con�
trol extracts (from leaves infiltrated with bacteria
carrying pICH6692 only) was subtracted from val�
ues of GFP containing crude protein extracts. The
identity of GFP in the extracts to the standard was
proved by recording their fluorescence spectra.
The concentration of total soluble protein was
determined by the method of Bradford [18].
The SDS�PAGE analyses of protein extracts
with Coomassie staining were carried out accord�
ing to Blakesly and Boezi [19]. Crude protein
extracts were mixed with 3 X loading buffer, boiled
during 5 min and applied on 12 % gel.
ISSN 0564–3783. Цитология и генетика. 2008. № 218
Y.R. Sindarovska, Y.V. Sheludko, I.M. Gerasymenko, M.A. Bannikova, N.V. Kuchuk
For determination of relative abundance of
GFP band on Coomassie stained SDS gel we used
Gel�Pro Analyzer (v. 3.1.00.00, Media Cyberne�
tics). Background correction: Join valleys with
maximal slope 20.
Results and discussion. Transient expression of
GFP in N. excelsior. In our recent publication we
showed that as a host for Agrobacterium�mediated
transient expression N. excelsior displayed the best
characteristics in regard to biomass yield as well as
GFP accumulation level for different types of the
expression cassettes. For preparative production of
recombinant GFP 2nd–4th upper leaves of green�
house grown plants of N. excelsior were filled with
Agrobacterium suspensions harboring different
plasmid vectors mixed in equal volumes (viral�
based module system which was described in
details in the recent publications [7, 13, 14]). The
p19 protein of tomato bushy stunt virus, a suppres�
sor of post�translational gene silencing, was co�
expressed with the GFP gene to increase the
reporter protein accumulation [18].
In order to confirm that the effectiveness of the
purification scheme does not depend on the high
initial recombinant protein level we performed
transient expression under conditions which were
not optimal for the maximal GFP accumulation
(plant developmental stage and cultivation temper�
ature [7]). Leaf tissue of the infiltrated plants was
harvested and extracted at the 18th day after infiltra�
tion. GFP content in the crude protein extract
amounted to 3.3 % of the total soluble protein.
Protein purification scheme. For extraction of
GFP from leaf tissue we used a buffer without
additional protectors against proteases because
GFP proteolysis resistance was described recently
[10]. As the first purification stage we selected pre�
cipitation with ammonium sulfate. Ammonium
sulfate precipitates cellular debris, ribosomes, and
membrane fragments and stabilizes most of the
proteins in solution. This stage has been used in
several GFP purification schemes [10, 11, 20]. In
our experiments we initially performed stepwise
ammonium sulfate precipitation of GFP from
crude protein extract in order to determine the
optimal concentration range. We observed the
highest GFP concentration in the fractions bet�
ween 50 % (app. 2.3 M) and 70 % (app. 3 M) of
ammonium sulfate saturation (Fig. 1). These data
are in agreement with other protocols which have
Fig. 1. GFP precipitation with ammonium sulfate
Fig. 2. Protein fractionation by Q�Sepharose chromato�
graphy. Solid line – protein concentration, µg/mL; dashed
line – GFP fluorescence (Relative Units); dotted line –
buffer C concentration, �5 %; gray rectangle indicates col�
lected fractions
Fig. 3. SDS�PAGE analysis of protein fractions (Coomassie
staining): 1 – molecular weight marker proteins, kDa; 2 –
fractions after ammonium sulfate precipitation; 3–5 – frac�
tions collected after Q�Sepharose chromatography. GFP
position is indicated with arrow
ІSSN 0564–3783. Цитология и генетика. 2008. № 2 19
Purification of recombinant GFP produced by Agrobacterium(mediated
used salting�out conditions to fractionate GFP. For
example, range 40–70 % of ammonium sulfate
saturation was applied as an initial GFP purifica�
tion step from E. coli protein extract [10].
At the next step we used anion�exchange chro�
matography. The dialyzed protein extract was
applied onto a Q�Sepharose FF column. After
washing the column, the proteins were eluted with
a linear NaCl gradient and the highest GFP con�
centrations were observed at the fractions collect�
ed from 75 to 95 mL after start of chromatography
(35–55 mL after start of elution that corresponds
to app. 0.14–0.25 M NaCl concentration in the
buffer, respectively) (Fig. 2).
The concentration of GFP in fractions during
purification procedure was estimated by measure�
ment of fluorescence intensity and comparison with
standard values. The background fluorescence of the
initial crude protein extract (from leaves infiltrated
with bacteria carrying pICH6692 only) was subtract�
ed from values of initial GFP containing extract.
The developed scheme of enrichment resulted
in 26�fold purification of the recombinant GFP
(Table). GFP was recovered in high (77 %) yield
with about 85 % purity as it was calculated by
comparison of GFP containing fraction fluores�
cence with the standard fluorescence means.
The collected GFP containing fractions were
additionally tested with SDS PAGE (Fig. 3) and
relative abundance of GFP band on Coomassie
stained gel was estimated by densitometry analysis
using Gel Pro Analyzer software algorithm. The
obtained results are in good agreement with those
calculated by fluorescence measurements: the
average GFP homogeneity in the collected frac�
tions was 85.1 % reaching in several fractions more
then 90 %.
In conclusion, we developed the efficient pro�
tocol for production and purification of recombi�
nant GFP from plant source using Agrobacterium�
mediated transient expression.
The developed protein purification scheme
included ammonium sulfate precipitation and Q�
sepharose anion�exchange chromatography and
integrates effective purification (85 % homogeni�
ty) with high protein yield (about 75 % of initial
GFP content).
The described purification procedure was per�
formed in gentle environment conditions (in com�
parison with several other protocols including
organic solvent extraction [10] or high tempera�
ture precipitation [20]) and, although some opti�
mization may be required, we consider this scheme
may be regarded as a benchmark for purifying of
GFP�fusion proteins as well as GFP.
Acknowledgements. The authors are grateful to the
Ministry for Education and Science of Ukraine and
National Academy of Science (grant DNTP 3.2.
DP/148–2003 and 15K�2004) for financial support.
РЕЗЮМЕ. Зелений флуоресцентний білок (GFP)
часто використовують як репортерний білок у різних
галузях біологічних досліджень. Ефективний протокол
Agrobacterium�опосередкованої транзієнтної експресії в
Nicotiana excelsior було використано для швидкого одер�
жання препаративної кількості рекомбінантного GFP.
Розроблено схему очищення рекомбінантного білка,
яка включає стадію осадження сульфатом амонію
та іонообмінну хроматографію на сорбенті Q�sepharose.
В результаті очищення було отримано фракцію білків
з вмістом GFP близько 85 %. В ході очищення було от�
римано близько 75 % від початкової кількості рекомбі�
нантного GFP.
РЕЗЮМЕ. Зеленый флюоресцентный белок (GFP)
часто используется в качестве маркерного белка в разных
областях биологических исследований. Эффективный
протокол Agrobacterium�опосредованной транзиентной
экспрессии в Nicotiana excelsior был использован для
быстрого получения препаративных количеств реком�
бинантного GFP. Разработана схема очистки рекомби�
нантного белка, включающая стадию осаждения суль�
фатом аммония и ионообменную хроматографию
на сорбенте Q�sepharose. В результате очистки была по�
лучена фракция белков с содержанием GFP около
85 %. В ходе очистки было выделено около 75 % от на�
чального количества рекомбинантного GFP.
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Y.R. Sindarovska, Y.V. Sheludko, I.M. Gerasymenko, M.A. Bannikova, N.V. Kuchuk
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Received 20.09.07
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