Development of the chromatographic medium for the affinity isolation of the recombinant hIFN-β1b based on immobilized single-chain antibodies
Aim. The development of a laboratory method for the production of immunoaffinity chromatography medium for the purification of the recombinant human IFN-β1b. Methods. A gene of the chimeric protein ScFvbINF-CBD was constructed using the DNA sequences encoding ScFv specific to hIFN-β1b and the cellul...
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| Cite this: | Development of the chromatographic medium for the affinity isolation of the recombinant hIFN-β1b based on immobilized single-chain antibodies / Ia.O. Pokholenko, O.B. Gorbatiuk, O.V. Okunev, D.M. Irodov, M.I. Degtiarova, O.G. Palivoda, V.A. Kordium // Вiopolymers and Cell. — 2015. — Т. 31, № 4. — С. 279-284. — Бібліогр.: 14 назв. — англ. |
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Pokholenko, Ia.O. Gorbatiuk, O.B. Okunev, O.V. Irodov, D.M. Degtiarova, M.I. Palivoda, O.G. Kordium, V.A. 2019-06-12T12:23:31Z 2019-06-12T12:23:31Z 2015 Development of the chromatographic medium for the affinity isolation of the recombinant hIFN-β1b based on immobilized single-chain antibodies / Ia.O. Pokholenko, O.B. Gorbatiuk, O.V. Okunev, D.M. Irodov, M.I. Degtiarova, O.G. Palivoda, V.A. Kordium // Вiopolymers and Cell. — 2015. — Т. 31, № 4. — С. 279-284. — Бібліогр.: 14 назв. — англ. 0233-7657 DOI: http://dx.doi.org/10.7124/bc.0008EC https://nasplib.isofts.kiev.ua/handle/123456789/152564 577.29: 57.088.3: 543.544.17 Aim. The development of a laboratory method for the production of immunoaffinity chromatography medium for the purification of the recombinant human IFN-β1b. Methods. A gene of the chimeric protein ScFvbINF-CBD was constructed using the DNA sequences encoding ScFv specific to hIFN-β1b and the cellulose-binding domain (CBD) of Clostridium thermocellum. The developed chimeric protein was expressed in Escherichia coli cells. The target protein was obtained in soluble, functionally active form by its renaturation from the bacterial inclusion bodies in vitro. The ScFvbINF-CBD was immobilized on a chitin carrier. Results. The introduction of CBD by gene engineering techniques enabled the oriented non-covalent immobilization of ScFvbINF-CBD on chromatographic matrix. The developed immunoaffinity medium allowed isolating the rhIFN-β1b from the complex mixture, after its renaturation from the inclusion bodies, with more than 89 % purity. Conclusion. The designed immunoaffinity medium provides isolating the rhIFN-β1b from the complex protein mixtures. Мета. Розробити лабораторний метод створення імуноафінного хроматографічного сорбенту для виділення та очищення рекомбінантного IFN-β1b людини. Методи. Використовуючи послідовності ДНК, які кодують ScFv специфічні до hIFN-β1b, та целюлозозв’язувальний домен (CBD) Clostridium thermocellum, створено ген химерного протеїну ScFvbINF-CBD. Проведено експресію створеного химерного протеїну в клітинах Escherichia coli. Цільовий білок одержували у розчинній, функціонально активній формі шляхом ренатурації з бактеріальних тілець включення in vitro. ScFvbINF-CBD іммобілізували на хітиновому носії. Результати. Введення CBD до складу химерного білка забезпечує орієнтовану, не ковалентну іммобілізацію ScFvbINF-CBD на хроматографічній матриці. За допомогою створеного імуноафінного сорбенту було виділено rhIFN-β1b зі складної суміші білків, одержаної після його ренатурації з тілець включення, з чистотою більше ніж 89 %. Висновки. Створений імуноафінний хроматографічний сорбент дозволяє виділяти рекомбінантний IFN-β1b людини зі складних сумішей білків. Цель. Разработать лабораторный метод создания иммуноаффинного хроматографического сорбента для выделения и очистки рекомбинантного IFN-β1b человека. Методы. Используя последовательности ДНК, кодирующие ScFv специфичные к hIFN-β1b, и целлюлозосвязывающий домен (CBD) Clostridium thermocellum, создан ген химерного белка ScFvbINF-CBD. Проведена экспрессия созданного химерного белка в клетках Escherichia coli. Целевой белок получали в растворимой, функционально активной форме путем ренатурации из бактериальных телец включения in vitro. ScFvbINF-CBD иммобилизовали на хитиновом носителе. Результаты. Введение CBD в состав химерного белка обеспечивает ориентированную не ковалентную иммобилизацию ScFvbINF-CBD на хроматографической матрице. С помощью созданного иммуносорбента был выделен rhIFN-β1b из сложной смеси белков, полученной после его ренатурации из телец включения, с чистотой более 89 %. Выводы. Созданный иммуноаффинный хроматографический сорбент позволяет выделять рекомбинатный IFN-β1b человека из сложных белковых смесей. This work was partially supplied by the grant «Developing a fundamental basis for obtaining immunoreagents of new generation». en Інститут молекулярної біології і генетики НАН України Вiopolymers and Cell Molecular and Cell Biotechnologies Development of the chromatographic medium for the affinity isolation of the recombinant hIFN-β1b based on immobilized single-chain antibodies Створення хроматографічного сорбенту на основі іммобілізованих одноланцюгових антитіл для афінного виділення рекомбінантного IFN-β1b людини Создание хроматографического сорбента основанного на иммобилизированных одноцепочечных антителах для аффинного выделения рекомбинантного IFN-β1b человека Article published earlier |
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Digital Library of Periodicals of National Academy of Sciences of Ukraine |
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DSpace DC |
| title |
Development of the chromatographic medium for the affinity isolation of the recombinant hIFN-β1b based on immobilized single-chain antibodies |
| spellingShingle |
Development of the chromatographic medium for the affinity isolation of the recombinant hIFN-β1b based on immobilized single-chain antibodies Pokholenko, Ia.O. Gorbatiuk, O.B. Okunev, O.V. Irodov, D.M. Degtiarova, M.I. Palivoda, O.G. Kordium, V.A. Molecular and Cell Biotechnologies |
| title_short |
Development of the chromatographic medium for the affinity isolation of the recombinant hIFN-β1b based on immobilized single-chain antibodies |
| title_full |
Development of the chromatographic medium for the affinity isolation of the recombinant hIFN-β1b based on immobilized single-chain antibodies |
| title_fullStr |
Development of the chromatographic medium for the affinity isolation of the recombinant hIFN-β1b based on immobilized single-chain antibodies |
| title_full_unstemmed |
Development of the chromatographic medium for the affinity isolation of the recombinant hIFN-β1b based on immobilized single-chain antibodies |
| title_sort |
development of the chromatographic medium for the affinity isolation of the recombinant hifn-β1b based on immobilized single-chain antibodies |
| author |
Pokholenko, Ia.O. Gorbatiuk, O.B. Okunev, O.V. Irodov, D.M. Degtiarova, M.I. Palivoda, O.G. Kordium, V.A. |
| author_facet |
Pokholenko, Ia.O. Gorbatiuk, O.B. Okunev, O.V. Irodov, D.M. Degtiarova, M.I. Palivoda, O.G. Kordium, V.A. |
| topic |
Molecular and Cell Biotechnologies |
| topic_facet |
Molecular and Cell Biotechnologies |
| publishDate |
2015 |
| language |
English |
| container_title |
Вiopolymers and Cell |
| publisher |
Інститут молекулярної біології і генетики НАН України |
| format |
Article |
| title_alt |
Створення хроматографічного сорбенту на основі іммобілізованих одноланцюгових антитіл для афінного виділення рекомбінантного IFN-β1b людини Создание хроматографического сорбента основанного на иммобилизированных одноцепочечных антителах для аффинного выделения рекомбинантного IFN-β1b человека |
| description |
Aim. The development of a laboratory method for the production of immunoaffinity chromatography medium for the purification of the recombinant human IFN-β1b. Methods. A gene of the chimeric protein ScFvbINF-CBD was constructed using the DNA sequences encoding ScFv specific to hIFN-β1b and the cellulose-binding domain (CBD) of Clostridium thermocellum. The developed chimeric protein was expressed in Escherichia coli cells. The target protein was obtained in soluble, functionally active form by its renaturation from the bacterial inclusion bodies in vitro. The ScFvbINF-CBD was immobilized on a chitin carrier. Results. The introduction of CBD by gene engineering techniques enabled the oriented non-covalent immobilization of ScFvbINF-CBD on chromatographic matrix. The developed immunoaffinity medium allowed isolating the rhIFN-β1b from the complex mixture, after its renaturation from the inclusion bodies, with more than 89 % purity. Conclusion. The designed immunoaffinity medium provides isolating the rhIFN-β1b from the complex protein mixtures.
Мета. Розробити лабораторний метод створення імуноафінного хроматографічного сорбенту для виділення та очищення рекомбінантного IFN-β1b людини. Методи. Використовуючи послідовності ДНК, які кодують ScFv специфічні до hIFN-β1b, та целюлозозв’язувальний домен (CBD) Clostridium thermocellum, створено ген химерного протеїну ScFvbINF-CBD. Проведено експресію створеного химерного протеїну в клітинах Escherichia coli. Цільовий білок одержували у розчинній, функціонально активній формі шляхом ренатурації з бактеріальних тілець включення in vitro. ScFvbINF-CBD іммобілізували на хітиновому носії. Результати. Введення CBD до складу химерного білка забезпечує орієнтовану, не ковалентну іммобілізацію ScFvbINF-CBD на хроматографічній матриці. За допомогою створеного імуноафінного сорбенту було виділено rhIFN-β1b зі складної суміші білків, одержаної після його ренатурації з тілець включення, з чистотою більше ніж 89 %. Висновки. Створений імуноафінний хроматографічний сорбент дозволяє виділяти рекомбінантний IFN-β1b людини зі складних сумішей білків.
Цель. Разработать лабораторный метод создания иммуноаффинного хроматографического сорбента для выделения и очистки рекомбинантного IFN-β1b человека. Методы. Используя последовательности ДНК, кодирующие ScFv специфичные к hIFN-β1b, и целлюлозосвязывающий домен (CBD) Clostridium thermocellum, создан ген химерного белка ScFvbINF-CBD. Проведена экспрессия созданного химерного белка в клетках Escherichia coli. Целевой белок получали в растворимой, функционально активной форме путем ренатурации из бактериальных телец включения in vitro. ScFvbINF-CBD иммобилизовали на хитиновом носителе. Результаты. Введение CBD в состав химерного белка обеспечивает ориентированную не ковалентную иммобилизацию ScFvbINF-CBD на хроматографической матрице. С помощью созданного иммуносорбента был выделен rhIFN-β1b из сложной смеси белков, полученной после его ренатурации из телец включения, с чистотой более 89 %. Выводы. Созданный иммуноаффинный хроматографический сорбент позволяет выделять рекомбинатный IFN-β1b человека из сложных белковых смесей.
|
| issn |
0233-7657 |
| url |
https://nasplib.isofts.kiev.ua/handle/123456789/152564 |
| citation_txt |
Development of the chromatographic medium for the affinity isolation of the recombinant hIFN-β1b based on immobilized single-chain antibodies / Ia.O. Pokholenko, O.B. Gorbatiuk, O.V. Okunev, D.M. Irodov, M.I. Degtiarova, O.G. Palivoda, V.A. Kordium // Вiopolymers and Cell. — 2015. — Т. 31, № 4. — С. 279-284. — Бібліогр.: 14 назв. — англ. |
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279
ISSN 0233-7657
Biopolymers and Cell. 2015. Vol. 31. N 4. P. 279–284
doi: http://dx.doi.org/10.7124/bc.0008EC
Molecular and Cell
Biotechnologies
UDC 577.29: 57.088.3: 543.544.17
Development of the chromatographic medium
for the affi nity isolation of the recombinant hIFN-β1b
based on immobilized single-chain antibodies
Ia. O. Pokholenko1, 2, O. B. Gorbatiuk1, 2, O. V. Okunev1, 2, D. M. Irodov1, 2,
M. I. Degtiarova1, O. G. Palyvoda1, 2, V. A. Kordium1, 2
1 Institute of Molecular Biology and Genetics, NAS of Ukraine
150, Akademika Zabolotnoho Str., Kyiv, Ukraine, 03680
2 State Institute of Genetic and Regenerative Medicine, NAMS of Ukraine
67, Vyshhorodska Str., Kyiv, Ukraine, 04114
yasnenka@gmail.com
Aim. The development of a laboratory method for the production of immunoaffi nity chromatography
medium for the purifi cation of the recombinant human IFN-β1b. Methods. A gene of the chimeric pro-
tein ScFvbINF-CBD was constructed using the DNA sequences encoding ScFv specifi c to hIFN-β1b and
the cellulose-binding domain (CBD) of Clostridium thermocellum. The developed chimeric protein was
expressed in Escherichia coli cells. The target protein was obtained in soluble, functionally active form
by its renaturation from the bacterial inclusion bodies in vitro. The ScFvbINF-CBD was immobilized on a
chitin carrier. Results. The introduction of CBD by gene engineering techniques enabled the oriented
non-covalent immobilization of ScFvbINF-CBD on chromatographic matrix. The developed immunoaffi n-
ity medium allowed isolating the rhIFN-β1b from the complex mixture, after its renaturation from the
inclusion bodies, with more than 89 % purity. Conclusion. The designed immunoaffi nity medium pro-
vides isolating the rhIFN-β1b from the complex protein mixtures.
K e y w o r d s: Interferon-β1b, single-chain antibodies, immunoaffi nity purifi cation, cellulose-binding
domain.
© 2015 Ia. Pokholenko et al.; Published by the Institute of Molecular Biology and Genetics, NAS of Ukraine on behalf of Biopolymers and Cell.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/),
which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited
Introduction
The purifi cation of a recombinant protein by immu-
noaffi nity chromatography is based on the interac-
tion of an antibody, which is covalently immobilized
on the matrix, with its antigen that separates the lat-
ter from the multicomponent mixture. The main ad-
vantage of this method is a potentially high selectiv-
ity allowing the isolation of trace quantities of a tar-
get product from the mixture. However, the genera-
tion of antibodies in bulk quantities, for the develop-
ment of such media, is a diffi cult and costly process
that requires the maintenance of either the signifi -
cant livestock of immunized animals or the hybrido-
ma cell line, which produces the antibodies specifi c
to the target antigen, and their further expression and
purifi cation. Special efforts also should be taken to
the elaboration of the isolated antibody immobiliza-
tion on the media, because the existing methods of
covalent immobilization (by crosslinking reagents
such as glutaraldehyde and etc.) are unable to pro-
vide the strictly oriented linkage of the antibody to
the matrix, and hence a signifi cant quantity of anti-
bodies loses their antigen binding properties owing
to the blocking or modifi cation of the antigen bind-
ing site of antibody, and etc. due to the immobiliza-
tion procedure. All facts mentioned above infl uence
both the technical characteristics of media, and their
280
Ia. O. Pokholenko, O. B. Gorbatiuk, O. V. Okunev et al.
cost. A technology of the recombinant single-chain
antibodies (ScFv), developed in the last decade, ena-
bles to simplify signifi cantly the process and reduce
the cost of obtaining antibodies. In addition, it is pos-
sible to introduce the DNA sequence for a protein,
which will provide oriented immobilization of ScFv
on a matrix, into the encoding sequence of the anti-
body by the genetic engineering techniques [1, 2].
The recombinant human interferon-β1b (rhIFN-
β1b) is the fi rst medicine approved for the treatment
of multiple sclerosis (MS). MS is an autoimmune
disease, which is characterized by the neurons de-
myelination in the central nervous system that leads
to the development of a wide spectrum of severe
neurological disorders [3]. Today, according to the
data presented by Multiple Sclerosis International
Federation [http://www.msif.org.], 2.3 million peo-
ple worldwide have MS and the number of patients
is continuously growing. The disease mainly affects
young people: from 25 to 40 years old. The thera-
peutic effect of rhIFN-β1b on the disease is attribut-
ed to its anti-infl ammatory properties, to the infl u-
ence on the endothelial cells, and on the permeability
of the blood brain barrier [4].
The present study is focused on the development
of the laboratory method for the production of im-
munoaffi nity chromatography medium for the puri-
fi cation of the rhIFN-β1b. The method is based on
the oriented non-covalent immobilization of the chi-
meric protein, which consists of ScFv against rhIFN-
β1b (ScFvbINF) and Clostridium thermocellum cellu-
lose-binding domain (CBD), on a chitin carrier.
Materials and Methods
ScFv against the rhIFN-β1b, obtained earlier at our
department from the plasmid vector pCANTAB-5E-
ScFv-β1INF, was subcloned into the plasmid vector
pET24-CBD-(Gly-spaser) via restriction sites Sfi I
and NotI. As a spacer for the spatial separation of
two affi nity centers of the chimeric protein (CBD
and ScFv) a sequence of 13 amino acids was intro-
duced [5, 6]. Professor Y. Shoham (Israel) kindly
provided the plasmid vector pCBD with the cellu-
lose binding domain (CBD) sequence from
Clostridium thermocellum for our research. The re-
sulting plasmid pET-24-ScFv-CBD was used for the
transformation of E. coli cells, BL21(DE3) strain.
For the expression of the target protein, an auto-in-
duction method described by Studier [7] was applied.
The localization and the content of the target protein in
the total lysate of E. coli cells were determined by elec-
trophoretic separation of the soluble and insoluble frac-
tions of the cell cytoplasmic proteins [8]. E.coli cells
were disrupted with lysozyme as described below [9].
The isolated inclusion bodies were solubilized in 20
mM Tris-HCl (pH 8.0), 7 M guanidine-HCl, 15 mM
2-mercaptoethanol for 1 h at room temperature and fi l-
tered through a 0.45 μm PVDF membrane fi lter (Mi-
llipore). ScFvbINF -CBD was purifi ed under denaturing
conditions using the Ni2+-immobilized affi nity chroma-
tography on Ni-NTA agarose (Qiagen).
Refolding of the purifi ed ScFvbINF – CBD was per-
formed by stepwise dilution [6]. After renaturation
ScFvbINF – CBD was immobilized on chitin beads
(New England Biolabs, UK). The resulting immu-
noaffi nity medium was washed with PBS pH 7.2, con-
taining 0.1 % Tween-20 and 0.14 M NaCl. Afterwards,
the rhIFN-1β renatured by stepwise dilution was ap-
plied to the column [5]. After washing of the immu-
noaffi nity medium with buffer PBS pH 7.2, contain-
ing 0.1 % Tween-20, 0.14 M NaCl to remove unbound
proteins, the rhIFN-β1b was eluted from the column
by decreasing pH of elution buffer: (0.1 M glycine and
0.5 M NaCl, pH 3.0). The rhIFN-β1b concentration in
the fraction eluted was quantifi ed by measuring ab-
sorbance of the solutions at 280 nm. The rhIFN-β1b
purity in the samples studied was determined by den-
sitometry of polyacrylamide gel electrophoregrams.
For this purpose the SDS-PAGE gels were stained
with Co omassie Brilliant Blue according to the manu-
facturers instructions, documented by ChemiDoc™
XRS+ Sy stem («Bio- Rad», USA), and subsequently
analyzed with «Image Lab Soft wareTM» («Bio-
Rad»).
Results and Discussion
1. Construction of chimeric protein
and expression in E. coli BL21 (DE3)
The CBD from the S1 subunit of the cellulolytic
281
Development of medium for isolation of rhIFN-β1b based on immobilized ScFv
complex of Clostridium thermocellum has been se-
lected as a partner protein for the fusion with the pre-
viously developed ScFv against the rhIFN-β1b [10].
It is worth mentioning, that the main features of the
domain are its ability to create a stable complex with
the hydrocarbon backbone of cellulose or chitin un-
der both native and denaturing conditions, and a high
effi ciency of the restoration of the molecule func-
tional structure after the renaturation from inclusion
bodies [11, 12]. After the subcloning, the E.coli cells
of the BL21(DE3) strain were transformed by the
developed pET24-ScFvbINF-CBD. It is known that the
components of the nutrient medium, the nature of
inducer substance, and the cultivation conditions
(temperature, aeration intensity, etc.), greatly infl u-
ence the level of expression of the recombinant pro-
teins in E.coli cells [13]. To enhance the yield of the
chimeric protein ScFvbINF-CBD we have tested the
infl uence of the complex nutrient medium for pro-
ducing strain BL21 (DE3)/pET 24–ScFvbINF-CBD on
the yield of the chimeric protein. Thus, the devel-
oped producing strain was cultivated on two differ-
ent media, namely LB and 2xYT. The expression of
ScFvbINF-CBD was induced by the addition of
α-lactose. It was shown that the cultivation on the
complex nutrient medium LB results in the synthesis
of the target chimeric protein with the expected mo-
lecular weight of 45 kDa, while the yield of the chi-
meric protein after cultivation in 2xYT was scarce
and hardly detectable. The accumulation of the tar-
get protein was 0.1 g out of 1 L of the bacterial cells
suspension in case of the LB medium. The study on
the distribution of the ScFvbINF-CBD in E. coli cells
demonstrated that it was accumulated in the inclu-
sion bodies, which were subsequently isolated after
the lysis of cells (Fig. 1.)
2. Purifi cation and renaturation
The isolated inclusion bodies were solubilized in the
buffer containing 7 M guanidine hydrochloride and
0.015 M 2-mercaptoethanol. After solubilization the
ScFvbINF-CBD was purifi ed by the metal ion affi nity
chromatography using the Ni-NTA agarose (Qiagen).
The purity of the obtained target protein was 95 % as
determined by the densitometry of polyacrylamide
gel electrophoregrams. The ScFvbINF-CBD was rena-
tured by step-wise dilution. The decrease of the con-
centration of guanidine hydrochloride was carried
out by step-wise dilution of the protein solution with
the renaturation buffer 20 mM Tris-HCl pH 8.0, 100
mM NaCl, 1mM EDTA in order to provide the fol-
lowing concentration levels of the chaotropic agent:
6 M→3 M → 2 M →1 M →0.5 M. At the concentra-
tions of 2 M →1 M →0.5 M the 0.4 M L-arginine
and the redox pair (GSH and GSSG) were added.
The GSH/GSSG pair catalyzes the disulfi de inter-
changes and thus ensures the correct folding of the
chimeric protein and the proper spatial arrangement
of both affi nity sites: CBD and the active site of
ScFvbINF. The renaturation effi ciency of the affi nity
sites was evaluated in accordance with their func-
tions: the ability of CBD to interact with the cellu-
lose carrier and the ScFv ability to bind rhIFN-β1b.
3. Immobilization of ScFvbINF-CBD
on chitin beads
It has been demonstrated that the affi nity interaction of
CBD with the cellulose or chitin leads to the formation
of the complex stabilized by the hydrophobic interac-
tion of certain amino acid residues and hydrocarbon
Fig. 1. Small-scale expression of the recombinant fusion protein
ScFv-(IFN-β)-CBD on LB. 1 – protein fractions from induced
BL21(DE3) cells carrying the plasmid pET-24-(IFN-β)-CBD; 2
– isolated inclusion bodies; 3 – cytoplasmic soluble fraction; 4
– molecular weight marker
1 2 3 4
116.0
66.2
45.0
35.0
25.0
18.4
14.4
282
Ia. O. Pokholenko, O. B. Gorbatiuk, O. V. Okunev et al.
backbone of the matrix [14]. The chitin beads have
been selected as the solid phase for immobilization of
the chimeric protein. The protein solution obtained af-
ter the renaturation procedure, was added to the slurry
of chitin beads, equilibrated with buffer 0.1 M Tris-HCl
(pH 8.0) containing 0.1 M NaCl, and incubated with
continuous agitation at 22 C for 2 hours. Thereafter the
medium was packed in the column and washed by 5
volumes of PBS pH 7.2, in order to wash away the un-
bound proteins. The proteins attached to the chitin car-
rier were eluted under denaturing conditions and sepa-
rated by SDS-PAGE. The analysis of electrophoregram
revealed the presence of chimeric protein in eluate. The
binding capacity of the chitin beads for the target pro-
tein was ~0.6–1 mg of the ScFvbINF-CBD/mL of the
medium as determined by the densitometry of poly-
acrylamide gel electrophoregrams.
4. Purifi cation of rhIFN-β1b
on developed medium
The solution of the rhIFN-β1b (obtained after its re-
naturation) containing a signifi cant amount of E.coli
proteins was applied of the column with developed
affi nity medium. The rhIFN-β1b was eluted by the
pH change: from pH 7.2 to pH 3.0. The analysis of
electrophoregrams obtained after the separation of
eluted proteins by SDS-PAGE (Fig. 2) revealed that
the purity of the rhIFN-β1b, eluted under conditions
studied, was 89 %, and the additional minor band
was also detected. Desorption of the ScFvifnb-CBD
from the medium was not observed under the condi-
tions studied. The data obtained by western-blot
analysis (Fig. 2.b) revealed that the additional band
with the molecular weight ~ 36 kDa represented the
dimer of rhIFN-β1b, originated from its oligomeri-
zation during renaturation. It should be noted that
the affi nity media based on the use of antibodies do
not allow separating the monomer from the oligomer
forms of the same protein in case of the preservation
of the epitope to which the antibody is specifi c. The
fi nal polishing of the eluate containing the rhIFN-
β1b from its oligomer forms can be carried out by
Fig. 2. Purifi cation of rhIFN-β on immunoaffi nity medium. Chromatogram (A): 1 – non-bound proteins washed from the column
with the buffer, 2 – rhIFN-β eluted from the column. Electrophoregram (B): 1-refolded rhIFN-β applied to the column, 2 – purifi ed
rhIFN-β eluted from the column under acidic conditions, 3 – immunoblotting of purifi ed rhIFN-β with polyclonal anti-rhIFN-β anti-
bodies (Sigma), M – molecular weight marker top to bottom: 116.0, 62.0, 45.0, 35.0, 25.0, 18.4, 14.4 kDa)
A B
1 2 3 М
rhIFN-β dimer
101.0
1
2
0.8
0.6
0.4
0.2
8
6
4
pH
mAU
pH
2 rhIFN-β
280 nm, mAU
0 12 24 V, ml
283
Development of medium for isolation of rhIFN-β1b based on immobilized ScFv
the fractionation of the protein mixture by a size-
exclusion chromatography on Superdex 75 10/300
GL column.
Concluding Remarks
The present study is focused on the development of the
laboratory method for obtaining the immunoaffi nity
medium for the chromatographic purifi cation of the
rhIFN-β1b. The proposed method is based on the ori-
ented non-covalent immobilization of the chimeric pro-
tein, consisting of the ScFv specifi c to the rhIFN-β1b
and the CBD from Clostridium thermocellum, on the
chitin carrier. The developed immunoaffi nity medium
allows isolating the rhIFN-β1b from the complex pro-
tein mixture after its renaturation from the inclusion
bodies, and purifi cation to 89 %. It is noteworthy that
the developed media does not separate the oligomeric
forms of rhIFN-β1b, appeared during the renaturation.
Acknowledgements
The authors thank Professor Y. Shoham, Dr. M. Pav-
lova, and Dr. P. Gilchuk for their valuable assistance
in the publication of the current work.
Funding
This work was partially supplied by the grant «De-
veloping a fundamental basis for obtaining immu-
noreagents of new generation».
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Створення хроматографічного сорбенту
на основі іммобілізованих одноланцюгових антитіл
для афінного виділення рекомбінантного
IFN-β1b людини
Я. О. Похоленко, О. Б. Горбатюк,
О. В. Окунєв, Д. М. Іродов, М. І. Дегтярьова,
О. Г. Паливода, В. А. Кордюм
Мета. Розробити лабораторний метод створення імуноафін-
ного хроматографічного сорбенту для виділення та очищен-
ня рекомбінантного IFN-β1b людини. Методи. Викорис то-
ву ючи послідовності ДНК, які кодують ScFv специфічні до
hIFN-β1b, та целюлозозв’язувальний домен (CBD) Clostri di-
um thermocellum, створено ген химерного протеїну ScFvbINF-
CBD. Проведено експресію створеного химерного протеїну
в клітинах Escherichia coli. Цільовий білок одержували у
розчинній, функціонально активній формі шляхом ренату-
рації з бактеріальних тілець включення in vitro. ScFvbINF-
CBD іммобілізували на хітиновому носії. Результати. Вве-
дення CBD до складу химерного білка забезпечує орієнто-
вану, не ковалентну іммобілізацію ScFvbINF-CBD на хромато-
графічній матриці. За допомогою створеного імуноафінного
284
Ia. O. Pokholenko, O. B. Gorbatiuk, O. V. Okunev et al.
сорбенту було виділено rhIFN-β1b зі складної суміші білків,
одержаної після його ренатурації з тілець включення, з чи-
стотою більше ніж 89 %. Висновки. Створений імуноафін-
ний хроматографічний сорбент дозволяє виділяти рекомбі-
нантний IFN-β1b людини зі складних сумішей білків.
Ключов і слова: інтерферон-β1b, одноланцюгові ан ти ті-
ла, імуноафінне очищення, целюлозозв’язувальний домен.
Создание хроматографического сорбента
основанного на иммобилизированных
одноцепочечных антителах для аффинного выделения
рекомбинантного IFN-β1b человека
Я. А. Похоленко, О. Б. Горбатюк,
О. В. Окунев, Д. М. Иродов, М. И. Дегтярева,
О. Г. Паливода, В. А. Кордюм
Цель. Разработать лабораторный метод создания иммуно-
аффинного хроматографического сорбента для выделения
и очистки рекомбинантного IFN-β1b человека. Методы. Ис-
по льзуя последовательности ДНК, кодирующие ScFv спе-
ци фичные к hIFN-β1b, и целлюлозосвязывающий домен
(CBD) Clostridium thermocellum, создан ген химерного бел-
ка ScFvbINF-CBD. Проведена экспрессия созданного химер-
ного белка в клетках Escherichia coli. Целевой белок полу-
чали в растворимой, функционально активной форме пу-
тем ренатурации из бактериальных телец включения in
vitro. ScFvbINF-CBD иммобилизовали на хитиновом носите-
ле. Результаты. Вве дение CBD в состав химерного белка
обеспечивает ориентированную не ковалентную иммоби-
лизацию ScFvbINF-CBD на хроматографической матрице. С
помощью созданного иммуносорбента был выделен rhIFN-
β1b из сложной смеси белков, полученной после его рена-
турации из телец включения, с чистотой более 89 %. Вы-
воды. Созданный иммуноаффинный хроматографический
сорбент позволяет выделять рекомбинатный IFN-β1b чело-
века из сложных белковых смесей.
Ключевые слова: интерферон-β1b, одноцепочечные ан-
титела, иммуноаффинная очистка, целлюлозосвязывающий
домен.
Received 01.05.2015
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