Comparison of different systems for purification of recombinant proteins produced by transient expression in plants
Three different approaches for purification of transiently expressed recombinant proteins from plants using GFP as a reporter have been successfully applied. The purification scheme including ammonium sulfate precipitation and anion-exchange chromatography was compared with two tag-based protocol...
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| Опубліковано в: : | Фактори експериментальної еволюції організмів |
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| Дата: | 2008 |
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Інститут молекулярної біології і генетики НАН України
2008
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| Назва журналу: | Digital Library of Periodicals of National Academy of Sciences of Ukraine |
| Цитувати: | Comparison of different systems for purification of recombinant proteins produced by transient expression in plants / N.M. Lypova, Y.R. Sindarovska, I.M. Gerasymenko, Y.V. Sheludko, M.A. Bannikova, N.V. Kuchuk // Фактори експериментальної еволюції організмів: Зб. наук. пр. — 2008. — Т. 5. — С. 397-400. — Бібліогр.: 10 назв. — англ. |
Репозитарії
Digital Library of Periodicals of National Academy of Sciences of Ukraine| _version_ | 1859520464420864000 |
|---|---|
| author | Lypova, N.M. Sindarovska, Y.R. Gerasymenko, I.M. Sheludko, Y.V. Bannikova, M.A. Kuchuk, N.V. |
| author_facet | Lypova, N.M. Sindarovska, Y.R. Gerasymenko, I.M. Sheludko, Y.V. Bannikova, M.A. Kuchuk, N.V. |
| citation_txt | Comparison of different systems for purification of recombinant proteins produced by transient expression in plants / N.M. Lypova, Y.R. Sindarovska, I.M. Gerasymenko, Y.V. Sheludko, M.A. Bannikova, N.V. Kuchuk // Фактори експериментальної еволюції організмів: Зб. наук. пр. — 2008. — Т. 5. — С. 397-400. — Бібліогр.: 10 назв. — англ. |
| collection | DSpace DC |
| container_title | Фактори експериментальної еволюції організмів |
| description | Three different approaches for purification of transiently expressed recombinant
proteins from plants using GFP as a reporter have been successfully applied. The purification
scheme including ammonium sulfate precipitation and anion-exchange chromatography was
compared with two tag-based protocols applying metal affinity chromatography with a 6xHis
tag and intein mediated purification with a chitin-binding affinity tag.
Три різних підходи для очищення рекомбінантних білків, отриманих методом
транзієнтної експресії в рослинах, було успішно застосовано з використанням GFP як
репортера. Схему очищення, яка включала преципітацію сульфатом амонію і
аніонообмінну хроматографію, порівнювали з протоколами тегового очищення з
використанням 6xHis-тега та хітин-інтеін-опосередкованого очищення.
Три различных подхода для очистки рекомбинантных белков, полученных
методом транзиентной экспрессии в растениях, были успешно применены с
использованием GFP как репортера. Схему очистки, включающую преципитацию
сульфатом аммония и анионообменную хроматографию, сравнивали с протоколами
теговой очистки с использованием 6xHis-тега и хитин-интеин-опосредованной очистки.
|
| first_indexed | 2025-11-25T20:56:32Z |
| format | Article |
| fulltext |
397
4. Струговщикова Л.П., Шавловський Г.М., Федорович І.П., Кучерас Р.В.
Очищення та деякі властивості лужної фосфатази І дріжджів Pichia guilliermondi //
Укр. біохім. журн. – 1973. – Т. 45, № 3. – С. 312-317.
5. Шавловский Г.М., Жарова В.П., Щелокова И.Ф., Трач В.М., Сибирный А.А.,
Кшеминская Г.П. Флавиногенная активность природных штаммов дрожжей Pichia
guilliermondii // Прикл. биохимия и микробиология. – 1978. – Т.14. – С. 184-189.
6. Bessey O.A., Lowry J.Y., Love R.H.J. The fluorometric measurement of the
nucleotides of riboflavin and their concentration in tissues // J. Biol. Chem. – 1949. – Vol.
180, N 2. – P. 378-381.
7. Lowry O.H., Rosebrough N.J., Farr A.L. and Randall R.J. Protein measurement
with the Folin phenol reagent // J. Biol. Chem. – 1951. – Vol. 193. – P. 265-275.
Резюме
Досліджено флавіногенну активність рекомбінантних штамів дріжджів Candida
famata, що містять ген РФ-кінази FMN1 під контролем сильного конститутивного
промотора TEF1. Оптимізовано умови нагромадження ФМН у культуральній рідині.
Изучена флавиногенная активность рекомбинантных штаммов дрожжей Candida
famata, содержащих ген РФ-киназы FMN1 под контролем сильного конститутивного
промотора TEF1. Оптимизированы условия накопления ФМН в культуральной
жидкости.
The flavinogenic activity of recombinant strains of the yeast Candida famata that
express the FMN1 gene encoding riboflavin kinase under control of the strong constitutive
TEF1 promoter was studied. Conditions for flavinmononucleotide production were optimized.
LYPOVA N.M., SINDAROVSKA Y.R, GERASYMENKO I.M., SHELUDKO Y.V.,
BANNIKOVA M.A., KUCHUK N.V.
Institute of Cell Biology and Genetic Engineering NAS of Ukraine,
Zabolotnogo str. 148, Kiev 03680, Ukraine, e-mail: ysheludko@ukr.net
COMPARISON OF DIFFERENT SYSTEMS FOR PURIFICATION OF
RECOMBINANT PROTEINS PRODUCED BY TRANSIENT EXPRESSION IN
PLANTS
Plants as source of recombinant proteins have important advantages over microbial or
animal cell systems. Plant cells, unlike bacteria, are able to produce proteins with post-
translational modifications, as well as correctly folded and assembled multimeric proteins, e.
g. antibodies [1]. In contrast to animal cells, plants are free from human pathogens like
viruses and prions, so the recombinant proteins of plant origin are considered to be safer [2].
The main obstacle on the way of using transgenic plants for high-scale production of
recombinant proteins is the low level of foreign gene expression in case of stable integration
into plant nuclear genome (usually about 0.1-0.5 % TSP) [3]. Transient gene expression may
allow for rapid accumulation of considerably larger amount of recombinant proteins (in the
range 0.5-10 % TSP or sometimes more) [4], but even in this case an efficient purification
system can substantially improve the yield of pure target product.
Here we describe comparative analysis of different approaches for purification of
transiently expressed recombinant proteins from plants using green fluorescent protein (GFP)
as a reporter. The purification scheme including ammonium sulfate precipitation and anion-
exchange chromatography was compared with two tag-based protocols applying metal
affinity chromatography with a 6xHis tag (Qiagen) and intein mediated purification with a
chitin-binding affinity tag (New England Biolab).
398
Materials and methods
For transient expression we used a 35S expression system carrying the GFP gene
driven by the CaMV 35S promoter (pICH5290) and a viral-based expression system
(pICH10881, pICH10570 and pICH7410 ) carrying the same reporter gene described in
details in the recent publications [5-7]). In all experiment p19 protein of Tomato bushy stunt
virus, a suppressor of post-transcriptional gene silencing, was co-expressed (pICH6692) [8].
For tag-based purification, either the sequence coding for six histidine residues or the
fragment encoding intein and chitin-binding domain (derived from the pTYB1 plasmid, New
England Biolabs) was fused to the GFP gene immediately before the stop-codon.
Infiltration of Nicotiana excelsior plants with Agrobacterium tumefaciens strain
GV3101 and transient expression was performed as it was described [7]. Leaf tissue was
extracted with 100 mM KPi buffer, pH 7.8, containing 5 mM EDTA and 10 mM β-
mercaptoethanol. The protein fraction precipitated between 50 % and 70 % of ammonium
sulfate saturation was separated by anion-exchange chromatography on Q-sepharose with a
linear NaCl gradient (0-1 M NaCl) [9]. For purification of the His-tagged GFP, the leaf
extracts (prepared in 50 mM Tris/HCl buffer, pH 8.0, containing 300 mM NaCl, 10 mM
imidazole, 10 mM β-mercaptoethanol, and 2.5 % PVP) were applied onto Ni-NTA sorbent,
the column was washed with extraction buffer and the target protein was eluted with the same
buffer containing 250 mM imidazole. For intein-mediated purification, the leaf extracts
(prepared in 100 mM KPi buffer, pH 7.8, containing 5 mM EDTA, 10 mM β-
mercaptoethanol, and 2.5 % PVP) were applied onto chitin sorbent, the column was washed
with extraction buffer and the target protein was eluted after intein cleavage induced by 50
mM dithiothreitol (DTT).
The content of GFP was calculated by measurements of fluorescence intensity in
dilutions of leaf extracts using fluorescence spectrophotometer Hitachi 4000 (Hitachi, Tokyo,
Japan) (excitation at 395 nm, emission at 509 nm) on the basis of standard values. The
background fluorescence of control extracts (from leaves infiltrated with bacteria carrying
pICH6692 only) was subtracted from values of GFP containing 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. The protein
mixtures were analyzed by SDS-PAGE with Coomassie staining.
Results and discussion
N. excelsior was used in our experiments because it was shown to be a promising host
for production of recombinant proteins by means of Agrobacterium-mediated transient
expression [7]. The GFP gene was expressed under control of CaMV 35S promotor or as a
part of a module viral-based system which allows considerable increase of the reporter protein
accumulation [5, 6]. However, in order to confirm that the effectiveness of the purification
scheme does not depend on 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 temperature [9]). GFP content in the crude protein
extract amounted to 3.3 % TSP. The crude protein extract from infiltrated leaf tissue was
subjected to ammonium sulfate precipitation followed by Q-sepharose anion-exchange
chromatography. The concentration of GFP in fractions during purification procedure was
estimated by measurements of fluorescence intensity. The developed scheme of enrichment
resulted in 26-fold purification of the recombinant GFP. GFP was recovered in high yield (77
%) with about 85 % purity. The described purification procedure was performed in gentle
environment conditions and, although some optimization may be required, we consider this
scheme may be regarded as a benchmark for purifying of GFP-fusion proteins as well as GFP.
After cloning from the jellyfish Aequorea victoria the GFP gene has undergone
substantial modifications that resulted in high expression rate, increased fluorescence,
stability and low toxicity for wide range of hosts, including plant cells [10]. Such
modifications are not always desirable for pharmaceutically valuable proteins, thus their level
399
of accumulation in plants is often considerably lower than that of GFP. It renders classical
purification schemes ineffective. Adding of affinity tags to the protein of interest simplifies
the purification procedure and improves the yield of the refined recombinant protein. Proteins
tagged with 6 consecutive histidine residues can be efficiently purified using nickel-
nitrilotriacetic acid (Ni-NTA) metal-affinity chromatography matrix (Qiagen).
The GFP gene with additional sequence coding for six histidine residues was
successfully transiently expressed in N. excelsior under control of CaMV 35S promoter. The
level of His-tagged GFP reached 5.5 % TSP, which is normal mean for such kind of
expression system [7]. One-stage purification procedure resulted in obtaining of homogenous
target protein (Fig. 1), although the yield of GFP was about 40 %. The effectiveness of His-
tag-mediated purification was shown for many recombinant proteins in different host systems,
but for production of pharmaceutically valuable proteins in plants this method has some
drawbacks. The protease cleavage of His-tag, which is necessary for obtaining of native
product, is an expensive procedure. In addition, some plant proteins containing histidine
residues can interact with Ni-NTA matrix and pollute the target product. To overcome these
problems, other affinity tag-based systems may be used.
IMPACT (Intein Mediated Purification with an Affinity Chitin-binding Tag, New
England Biolabs) is a protein purification system which utilizes the inducible self-cleavage
activity of a protein splicing element (intein) to separate the target protein from the affinity
tag (chitin-binding domain). It allows purifying in a single chromagraphic step without further
protease cleavage a native recombinant protein. The drawback of this system is the lower
level of fused protein expression, presumably due to the big size of the tag. The GFP gene
with additional sequence coding for intein and chitin-binding domain was transiently
expressed in N. excelsior under control of CaMV 35S promoter. Although starting with
comparatively low accumulation level (about 1 % TSP), we were able to obtain homogenous
native GFP after one-step purification procedure (data not shown). Although further studies
are necessary for optimization of the expression protocol, we can conclude that IMPACT
system may be useful for purification of recombinant pharmaceutically valuable proteins
transiently expressed in plants in low amounts.
Conclusions
All three tested purification systems can be applied for obtaining of refined recombinant
GFP from plant tissues. After optimization they can be considered for purification of other
recombinant proteins produced in plants by means of Agrobacterium-mediated transient
expression.
References
1. Stoger E., Sack M., Fischer R., Christou P. Plantibodies: applications, advantages and
Figure 1. GFP-6xHis SDS-PAGE analyses: a) Coomassie
staining of proteins (all samples except of marker without prior
heat denaturation); 1 - molecular weight marker proteins, 2 –
crude protein extract of N. excelsior infiltrated with GFP-
6xHis; 3 – Ni-NTA purified protein; GFP is indicated with
arrow b) GFP fluorescence in protein extracts without prior
heat denaturation (under UV-light).
400
bottlenecks. // Curr. Opin. Biotechnol. – 2002. – Vol. 13. – P. 161-166.
2. Larrick J.W., Thomas D.W. Producing proteins in transgenic plants and animals. //
Curr. Opin. Biotechnol. – 2001. – Vol. 12. – P. 411-418.
3. Daniell H., Streatfield S.J., Wycoff K. Medical molecular farming: production of
antibodies, biopharmaceuticals and edible vaccines in plants. // Trends in Plant Sci. – 2001. –
Vol. 6. – P. 219–226.
4. Gleba Y., Klimyuk V. Marillonnet S. Magnifection – a new platform for expressing
recombinant vaccines in plants. // Vaccine. – 2005. – Vol. 23. – P. 2042–2048.
5. Marillonnet S., Giritch A., Gils M., Kanzia R., Klimyuk V., Gleba Y. In planta
engineering of viral RNA replicons: efficient assembly by recombination of DNA modules
delivered by Agrobacterium. // PNAS. – 2004. – №101. – P. 6852–6857.
6. Marillonnet S., Thoeringer C., Kandzia R., Klimyuk V., Gleba Y. Systemic
Agrobacterium tumefaciens-mediated transfection of viral replicons for efficient transient
expression in plants. // Nat. Biotechnol. – 2005. – Vol. 23. – P. 718–723.
7. Sheludko Y. V., Sindarovska Y. R., Gerasymenko I. M., Bannikova M. A., Kuchuk N. V.
Comparison of several Nicotiana species as hosts for high-scale Agrobacterium-mediated
transient expression. // Biotechnol. Bioeng. – 2007. – Vol. 96. – P. 608-614.
8. Voinnet O., Rivas S., Mestre P., Baulcombe D. An enhanced transient expression
system in plants based on suppression of gene silecing by the p19 protein of tomato bushy
stunt virus. // Plant J. – 2003. – Vol. 33. – P. 949–956.
9. Sindarovska Y. R., Sheludko Y. V., Gerasymenko I. M., Bannikova M. A., Kuchuk N. V.
Purifikation of recombinant GFP produced by Agrobacterium-mediated transient expression
in Nicotiana excelsior. // Cytology and Genetics. – Vol. 42 (N2). – (in press).
10. Chiu W., Niwa Y., Zeng W., Hirano T., Kobayashi H., Sheen J. Engineered GFP as a
vital reporter in plants. // Curr. Biol. – 1996. – Vol. 6. – P. 325–330.
Summary
Three different approaches for purification of transiently expressed recombinant
proteins from plants using GFP as a reporter have been successfully applied. The purification
scheme including ammonium sulfate precipitation and anion-exchange chromatography was
compared with two tag-based protocols applying metal affinity chromatography with a 6xHis
tag and intein mediated purification with a chitin-binding affinity tag.
Три различных подхода для очистки рекомбинантных белков, полученных
методом транзиентной экспрессии в растениях, были успешно применены с
использованием GFP как репортера. Схему очистки, включающую преципитацию
сульфатом аммония и анионообменную хроматографию, сравнивали с протоколами
теговой очистки с использованием 6xHis-тега и хитин-интеин-опосредованной очистки.
Три різних підходи для очищення рекомбінантних білків, отриманих методом
транзієнтної експресії в рослинах, було успішно застосовано з використанням GFP як
репортера. Схему очищення, яка включала преципітацію сульфатом амонію і
аніонообмінну хроматографію, порівнювали з протоколами тегового очищення з
використанням 6xHis-тега та хітин-інтеін-опосередкованого очищення.
|
| id | nasplib_isofts_kiev_ua-123456789-178358 |
| institution | Digital Library of Periodicals of National Academy of Sciences of Ukraine |
| issn | 2219-3782 |
| language | English |
| last_indexed | 2025-11-25T20:56:32Z |
| publishDate | 2008 |
| publisher | Інститут молекулярної біології і генетики НАН України |
| record_format | dspace |
| spelling | Lypova, N.M. Sindarovska, Y.R. Gerasymenko, I.M. Sheludko, Y.V. Bannikova, M.A. Kuchuk, N.V. 2021-02-18T15:40:18Z 2021-02-18T15:40:18Z 2008 Comparison of different systems for purification of recombinant proteins produced by transient expression in plants / N.M. Lypova, Y.R. Sindarovska, I.M. Gerasymenko, Y.V. Sheludko, M.A. Bannikova, N.V. Kuchuk // Фактори експериментальної еволюції організмів: Зб. наук. пр. — 2008. — Т. 5. — С. 397-400. — Бібліогр.: 10 назв. — англ. 2219-3782 https://nasplib.isofts.kiev.ua/handle/123456789/178358 Three different approaches for purification of transiently expressed recombinant proteins from plants using GFP as a reporter have been successfully applied. The purification scheme including ammonium sulfate precipitation and anion-exchange chromatography was compared with two tag-based protocols applying metal affinity chromatography with a 6xHis tag and intein mediated purification with a chitin-binding affinity tag. Три різних підходи для очищення рекомбінантних білків, отриманих методом транзієнтної експресії в рослинах, було успішно застосовано з використанням GFP як репортера. Схему очищення, яка включала преципітацію сульфатом амонію і аніонообмінну хроматографію, порівнювали з протоколами тегового очищення з використанням 6xHis-тега та хітин-інтеін-опосередкованого очищення. Три различных подхода для очистки рекомбинантных белков, полученных методом транзиентной экспрессии в растениях, были успешно применены с использованием GFP как репортера. Схему очистки, включающую преципитацию сульфатом аммония и анионообменную хроматографию, сравнивали с протоколами теговой очистки с использованием 6xHis-тега и хитин-интеин-опосредованной очистки. en Інститут молекулярної біології і генетики НАН України Фактори експериментальної еволюції організмів Біотехнології в медицині і сільському господарстві Comparison of different systems for purification of recombinant proteins produced by transient expression in plants Article published earlier |
| spellingShingle | Comparison of different systems for purification of recombinant proteins produced by transient expression in plants Lypova, N.M. Sindarovska, Y.R. Gerasymenko, I.M. Sheludko, Y.V. Bannikova, M.A. Kuchuk, N.V. Біотехнології в медицині і сільському господарстві |
| title | Comparison of different systems for purification of recombinant proteins produced by transient expression in plants |
| title_full | Comparison of different systems for purification of recombinant proteins produced by transient expression in plants |
| title_fullStr | Comparison of different systems for purification of recombinant proteins produced by transient expression in plants |
| title_full_unstemmed | Comparison of different systems for purification of recombinant proteins produced by transient expression in plants |
| title_short | Comparison of different systems for purification of recombinant proteins produced by transient expression in plants |
| title_sort | comparison of different systems for purification of recombinant proteins produced by transient expression in plants |
| topic | Біотехнології в медицині і сільському господарстві |
| topic_facet | Біотехнології в медицині і сільському господарстві |
| url | https://nasplib.isofts.kiev.ua/handle/123456789/178358 |
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