Cloning, expression and purification of D-Tyr-tRNATyr-deacylase from Thermus thermophilus
D-Tyr-tRNATyr-deacylase (DTD) is a conservative enzyme, found in all domains of life, which ensures an additional checkpoint in the recycling of misaminoacylated D-Tyr-tRNATyr. DTD is capable of accelerating the hydrolysis of the ester linkage of D-Tyr-tRNATyr producing a free tRNA and D-tyrosine, t...
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irk-123456789-1563422019-06-19T01:28:02Z Cloning, expression and purification of D-Tyr-tRNATyr-deacylase from Thermus thermophilus Rybak, M.Yu. Kovalenko, O.P. Kryklyvyi, I.A. Tukalo, M.A. Structure and Function of Biopolymers D-Tyr-tRNATyr-deacylase (DTD) is a conservative enzyme, found in all domains of life, which ensures an additional checkpoint in the recycling of misaminoacylated D-Tyr-tRNATyr. DTD is capable of accelerating the hydrolysis of the ester linkage of D-Tyr-tRNATyr producing a free tRNA and D-tyrosine, thereby preventing an incorrect incorporation of D-amino acids into proteins. Deacylase distinguishes between D- and L-aminoacyl moieties and does not hydrolyze L-aminoacylated tRNA. The structural bases of this specificity and the mechanism of D-aminoacyl-tRNA hydrolysis are poorly understood. Aim. To clone D-Tyr-tRNATyr-deacylase from T. thermophilus (DTDTT), optimize the conditions for its expression in E.coli and develop an efficient purification procedure yielding the high quality enzyme suitable for the structural and functional studies. Methods. For amplification of DTD gene from T. thermophilus genomic DNA and its cloning into the pProEXHTb expression vector modern techniques were applied. Purification of the recombinant DTD protein was done with three types of column chromatography. His-tag was cleaved out from DTD by TEV protease. The cleavage was confirmed by Western blot analysis with anti-His-tag antibodies. Molecular weight of purified DTDTT was determined by the gel-filtration. Results. The expression construct pProEXHTb, containing DTD sequence from T. thermophilus, was obtained and successfully expressed in the BL21(DE3)pLysS E.coli strain. The protein of interest was purified to homogeneity by the combination of affinity (Ni-NTA), anion-exchange (Q-Sepharose) and size-exclusion (Superdex S 200) chromatographies. 2 mg of more than 90% pure recombinant DTD can be obtained from 1 L of bacterial culture. Molecular weight of purified DTD from T. thermophilus was determined to be 32 kDa, suggesting its dimeric structure. Conclusions. The pProEXHTb expression vector can be used for expression of DTD from T. thermophilus. The preparative amounts of DTD can be obtained after the three-step chromatographic procedures and used for further functional and structural studies. D-Тир-тРНКТир-деацилаза (DTD) є консервативним білком, знайденим у всіх царствах живої природи, що забезпечує додатковий корегувальний етап гідролізу помилково аміноацильованих субстратів D-Тир-тРНКТир DTD здатна приcкорювати гідроліз ефірного зв’язку в комплексі D-Тир-тРНКТир, утворюючи вільну тРНК та D-Тир, таким чином попереджуючи включення D-амінокислот до білків. Деацилаза розрізняє D- та L-аміноацильні залишки в аміноацильованій тРНК та не гідролізує останні. Структурні основи такої специфічності та механізм гідролізу D-аміноацил-тРНК за участі DTD є недостатньо зрозумілими. Мета. Клонувати D-Тир-тРНКТир-деацилазу з T. thermophilus (DTDTT), оптимізувати умов її експресії в E. coli, розробити ефективний метод очищення з високим виходом високоякісного ферменту, для структурних та функціональних досліджень. Методи. Ампліфікацію та клонування в pProEXHTb DTD гена геномної ДНК T. thermophilus проведено за стандартними молекулярно-біологічними методами. Очистку рекомбінантного DTD білка проводено хроматографічно. Залишки гістидину відщеплено від DTD протеазою вірусу тютюну (TEV), ефективність реакції перевірено Вестерн-блот аналізом з анти-His-антитілами. Молекулярну масу очищеної DTDTT визначено гель-фільтрацією. Результати. Отримана конструкція pProEXHTb, що містить DTD послідовність з T. thermophilus, експресовано в E.coli штаму BL21(DE3)pLysS. Цільовий рекомбінантний білок виділено і очищено комбінацєю хроматографій: афінної (Ni-NTA), аніон-обмінної (Q-Sepharose) та гель-фільтрації (Superdex S 200) до чистоти 90%. Вихід рекомбінантної DTD склав 2 мг з одного літру культури. Визначена молекулярна маса очищеної DTD з T. thermophilus – 32 кДа свідчить на користь її димерної структури. Висновки. Отриманий вектор pProEXHTb, що містить DTD з T. thermophilus і підібрані умови очистки дозволяють отримати рекомбінантний DTD в кількостях достатніх для подальших структурно-функціональних досліджень. D-Тир-тРНКТир-деацилаза (DTD) является консервативным белком, найденным во всех царствах живой природы, обеспечивающим дополнительный корректирующий этап гидролиза ошибочно аминоацилированных субстратов D-Тир-тРНКТир. DTD ускоряет гидролиз эфирной связи в комплексе D-Тир-тРНКТир, предупреждая, таким образом, ошибочное включение D-аминокислот в белки. Деацилаза различает D- и L-аминоацильные остатки в аминоацилированной тРНК и не гидролизирует последние. Структурные основы такой специфичности и механизм гидролиза D-аминоацил-тРНК при участии DTD являются недостаточно изученными. Цель. Клонировать D-Тир-тРНКТир-деацилазу из T. thermophilus (DTDTT), оптимизировать условия её экспрессии в E. coli и разработать эффективный, с высоким выходом высококачественного фермента, метод очистки. Методы. Для амплификации и клонирования в pProEXHTb гена DTD геномной ДНК T. thermophilus использованы стандартные молекулярно-биологические методы. Очистку рекомбинантного DTD белка проводили хроматографически. Остатки гистидина отщепили протеазой вируса табака (TEV), еффективность реакции проверили Вестерн-блот анализом с анти-His-антителами. Молекулярный вес очищенной DTDTT определили гель-фильтрацией. Результаты. Полученная конструкция pProEXHTb, содержащая DTD последовательность T. thermophilus, экспрессирована в клетках E. coli штамма BL21(DE3)pLysS. Последовательными хроматографиями: афинной (Ni-NTA), анион-обменной (Q-Sepharose) и гель-фильтрации (Superdex S 200) получен гомогенный белок с чистотой более 90 %. Разработанная методика позволяет получить до 2 мг целевого белка / 1 л культуры. Определенный молекулярный вес очищенной DTD из T. thermophilus – 32 кДа свидетельствует о ее димерной структуре. Выводы. Полученная конструкция pProEXHTb, содержаащя DTD последовательность T. thermophilus и подобранные условия очистки позволяют получить рекомбинантный DTD для дальнейших структурно-функциональных исследований. 2015 Article Cloning, expression and purification of D-Tyr-tRNATyr-deacylase from Thermus thermophilus / M.Yu. Rybak, O.P. Kovalenko, I.A. Kryklyvyi, M.A. Tukalo // Вiopolymers and Cell. — 2015. — Т. 31, № 3. — С. 179-186. — Бібліогр.: 24 назв. — англ. 0233-7657 DOI: http://dx.doi.org/10.7124/bc.0008DE http://dspace.nbuv.gov.ua/handle/123456789/156342 577.217.32 en Вiopolymers and Cell Інститут молекулярної біології і генетики НАН України |
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Structure and Function of Biopolymers Structure and Function of Biopolymers |
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Structure and Function of Biopolymers Structure and Function of Biopolymers Rybak, M.Yu. Kovalenko, O.P. Kryklyvyi, I.A. Tukalo, M.A. Cloning, expression and purification of D-Tyr-tRNATyr-deacylase from Thermus thermophilus Вiopolymers and Cell |
| description |
D-Tyr-tRNATyr-deacylase (DTD) is a conservative enzyme, found in all domains of life, which ensures an additional checkpoint in the recycling of misaminoacylated D-Tyr-tRNATyr. DTD is capable of accelerating the hydrolysis of the ester linkage of D-Tyr-tRNATyr producing a free tRNA and D-tyrosine, thereby preventing an incorrect incorporation of D-amino acids into proteins. Deacylase distinguishes between D- and L-aminoacyl moieties and does not hydrolyze L-aminoacylated tRNA. The structural bases of this specificity and the mechanism of D-aminoacyl-tRNA hydrolysis are poorly understood. Aim. To clone D-Tyr-tRNATyr-deacylase from T. thermophilus (DTDTT), optimize the conditions for its expression in E.coli and develop an efficient purification procedure yielding the high quality enzyme suitable for the structural and functional studies. Methods. For amplification of DTD gene from T. thermophilus genomic DNA and its cloning into the pProEXHTb expression vector modern techniques were applied. Purification of the recombinant DTD protein was done with three types of column chromatography. His-tag was cleaved out from DTD by TEV protease. The cleavage was confirmed by Western blot analysis with anti-His-tag antibodies. Molecular weight of purified DTDTT was determined by the gel-filtration. Results. The expression construct pProEXHTb, containing DTD sequence from T. thermophilus, was obtained and successfully expressed in the BL21(DE3)pLysS E.coli strain. The protein of interest was purified to homogeneity by the combination of affinity (Ni-NTA), anion-exchange (Q-Sepharose) and size-exclusion (Superdex S 200) chromatographies. 2 mg of more than 90% pure recombinant DTD can be obtained from 1 L of bacterial culture. Molecular weight of purified DTD from T. thermophilus was determined to be 32 kDa, suggesting its dimeric structure. Conclusions. The pProEXHTb expression vector can be used for expression of DTD from T. thermophilus. The preparative amounts of DTD can be obtained after the three-step chromatographic procedures and used for further functional and structural studies. |
| format |
Article |
| author |
Rybak, M.Yu. Kovalenko, O.P. Kryklyvyi, I.A. Tukalo, M.A. |
| author_facet |
Rybak, M.Yu. Kovalenko, O.P. Kryklyvyi, I.A. Tukalo, M.A. |
| author_sort |
Rybak, M.Yu. |
| title |
Cloning, expression and purification of D-Tyr-tRNATyr-deacylase from Thermus thermophilus |
| title_short |
Cloning, expression and purification of D-Tyr-tRNATyr-deacylase from Thermus thermophilus |
| title_full |
Cloning, expression and purification of D-Tyr-tRNATyr-deacylase from Thermus thermophilus |
| title_fullStr |
Cloning, expression and purification of D-Tyr-tRNATyr-deacylase from Thermus thermophilus |
| title_full_unstemmed |
Cloning, expression and purification of D-Tyr-tRNATyr-deacylase from Thermus thermophilus |
| title_sort |
cloning, expression and purification of d-tyr-trnatyr-deacylase from thermus thermophilus |
| publisher |
Інститут молекулярної біології і генетики НАН України |
| publishDate |
2015 |
| topic_facet |
Structure and Function of Biopolymers |
| url |
http://dspace.nbuv.gov.ua/handle/123456789/156342 |
| citation_txt |
Cloning, expression and purification of D-Tyr-tRNATyr-deacylase from Thermus thermophilus / M.Yu. Rybak, O.P. Kovalenko, I.A. Kryklyvyi, M.A. Tukalo // Вiopolymers and Cell. — 2015. — Т. 31, № 3. — С. 179-186. — Бібліогр.: 24 назв. — англ. |
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Вiopolymers and Cell |
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179
UDC 577.217.32
Cloning, expression and purifi cation
of D-Tyr-tRNATyr-deacylase from Thermus thermophilus
M. Yu. Rybak, O. P. Kovalenko, I. A. Kryklyvyi, M. A. Tukalo
Institute of Molecular Biology and Genetics, NAS of Ukraine
150, Akademika Zabolotnoho Str., Kyiv, Ukraine, 03680
mariia.rybak@gmail.com
D-Tyr-tRNATyr-deacylase (DTD) is a conservative enzyme, found in all domains of life, which ensures an
additional checkpoint in the recycling of misaminoacylated D-Tyr-tRNATyr. DTD is capable of accelerating
the hydrolysis of the ester linkage of D-Tyr-tRNATyr producing a free tRNA and D-tyrosine, thereby prevent-
ing an incorrect incorporation of D-amino acids into proteins. Deacylase distinguishes between D- and L-
aminoacyl moieties and does not hydrolyze L-aminoacylated tRNA. The structural bases of this specifi city
and the mechanism of D-aminoacyl-tRNA hydrolysis are poorly understood. Aim. To clone D-Tyr-tRNATyr-
deacylase from T. thermophilus (DTDTT), optimize the conditions for its expression in E.coli and develop
an effi cient purifi cation procedure yielding the high quality enzyme suitable for the structural and func-
tional studies. Methods. For amplifi cation of DTD gene from T. thermophilus genomic DNA and its cloning
into the pProEXHTb expression vector modern techniques were applied. Purifi cation of the recombinant
DTD protein was done with three types of column chromatography. His-tag was cleaved out from DTD by
TEV protease. The cleavage was confi rmed by Western blot analysis with anti-His-tag antibodies. Molecu-
lar weight of purifi ed DTDTT was determined by the gel-fi ltration. Results. The expression construct
pProEXHTb, containing DTD sequence from T. thermophilus, was obtained and successfully expressed in
the BL21(DE3)pLysS E.coli strain. The protein of interest was purifi ed to homogeneity by the combination
of affi nity (Ni-NTA), anion-exchange (Q-Sepharose) and size-exclusion (Superdex S 200) chromatogra-
phies. 2 mg of more than 90% pure recombinant DTD can be obtained from 1 L of bacterial culture. Mo-
lecular weight of purifi ed DTD from T. thermophilus was determined to be 32 kDa, suggesting its dimeric
structure. Conclusions. The pProEXHTb expression vector can be used for expression of DTD from
T. thermophilus. The preparative amounts of DTD can be obtained after the three-step chromatographic
procedures and used for further functional and structural studies.
K e y w o r d s: D-amino acids, D-Tyr-tRNATyr-deacylase from T. thermophilus, cloning, expression, puri-
fi cation.
© 2015 M. Yu. Rybak 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
ISSN 0233-7657
Biopolymers and Cell. 2015. Vol. 31. N 3. P. 179–186
doi: http://dx.doi.org/10.7124/bc.0008DE
Introduction
D-amino acids are present in the cells of various spe-
cies from bacteria to mammals. In the bacterial walls
D-amino acids contribute to the resistance to proteo-
lytic digestion [1]. They also could be considered as
bacterial growth inhibitory factors that prevent a bio-
fi lm formation [2]. Furthermore, D-amino acids have
also been found in the proteins extracted from aged
human tissues [3]. These are the myelin basic pro-
tein, erythrocyte proteins, and L-amyloid pepti des
from Alzheimer disease brains [4]. D-amino acids
are shown to have toxic effects on the cells in both
prokaryotes and eukaryotes [5, 6, 7, 8].
Aminoacyl-tRNA-synthetases (aaRS), being spe-
cifi c to L-amino acids, ensure the fi rst step of D-ami-
no acids’ exclusion from protein synthesis. However,
the stereospecifi city of these enzymes is not abso-
lute: several aaRS have been found to charge tRNAs
with D-amino acids [9, 10]. D-Tyr-tRNATyr-deacyla-
180
M. Yu. Rybak, O. P. Kovalenko, I. A. Kryklyvyi, M. A. Tukalo
se is an enzyme responsible for the recycling of mis-
aminoacylated D-Tyr-tRNATyr, hydrolyzing an ester
bond between the amino acid and tRNA. However,
this enzyme has a broad specifi city [11], and may
accommodate different D-aminoacyl moieties, for
example, D-Tyr, D-Trp, D-Asp and D-Phe [9, 10].
The fi rst observations of the deacylase editing ac-
tivity in the extracts of E.coli, S.cerevisiae, rabbit
reticulocytes and rat liver were reported by Calendar
and Berg [9]. Later Soutourina et al. purifi ed D-Tyr-
tRNATyr-deacylase from E.coli [10, 12] and S.cerevi-
siae [10, 13]. Plant DTD was discovered as a product
of GEK1 gene that is involved in the ethanol toler-
ance in Arabidopsis thaliana [14]. The identifi cation
of DTD in other groups of organisms, including hu-
man [15], confi rms its widespread distribution in all
kingdoms of life and may be considered as an impor-
tant checkpoint of the translation machine specifi ci-
ty. In addition, the DTD amino acid sequences iden-
tity among prokaryotes and eukaryotes are highly
conservative [16, 17], suggesting a high conserva-
tive function of this enzyme in all living organisms.
Three classes of deacylases have been identifi ed:
class DTD1 has been found in most bacteria and all
eukaryotes [12], class DTD2 has been discovered in
archea and plants [14, 18], class DTD3 – in most
cyanobacteria [11]. The species with DTD1 have the
yihZ and dtd orthologous genes, responsible for the
deacylase activity. Despite the fact that the homo-
logues of dtd were found in different pro- and eu-
karyotic genomes, another type of DTD (dtd2) was
identifi ed in archea and subsequently in plants. In
contrast to the mainly dimeric DTD1 proteins, DTD2
has a monomer structure. In addition, the activity of
deacylases from the second class depends on the
presence of Zn2+ ions. The third type of D-Tyr-tRNATyr-
deacylases has been reported to be encoded by the
dtd3 gene (homologous to dtd1). DTD3 is a metal-
enzyme with two active sites for metal ions binding:
the fi rst one binds only Zn2+, the second – Ni2+, Mn2+
and Co2+ ions.
Some functional investigations of E.coli [12],
S. cerevisiae [10, 13], archaeal [18] D-Tyr-tRNATyr-
deacylases were performed, but profound structural
research that may explain the mechanism of D-aa-
tRNA hydrolysis by DTD, is still to be carried out. Un-
fortunately, the proposed catalytic mechanism of DTD
based on the crystal structures available at this time is
controversial and remains to be clarifi ed [17, 19].
In order to investigate the structural and function-
al properties of deacylase, one needs to have the pre-
parative quantities of this enzyme of a high purity. In
this work we describe the expression and purifi ca-
tion of DTD from T. thermophilus (DTDTT).
Materials and Methods
Cloning of DTDTT gene. Genomic DNA from T. ther-
mophilus cells were obtained according to [20]. Ba-
sed on the sequence information of the DTD gene from
T. thermophilus (152 amino acid residues) (http://
www.ncbi.nlm.nih.gov/protein/WP_01 1173028.1)
two PCR primers were designed Dtyr TT-N (5’-CCA
TGG CGG GCG GTG GTG CAG CGG GTC TCC)
and DtyrTT-C (5’-AAG CTT ATT AGC GTG GGC
GG AAG CGT TCC TCC GAG TCC). Sites for NcoI
and Hind III restriction enzymes were included in for-
ward and reverse primers, respecti vely. The primers
were previously phosphorylated by 10 unites of T4-
polynucleotide kinase («Thermo Scientifi c», Lithua-
nia) in the mixture containing 200 pmol of primer, 50
mM Tris-HCl (pH 7.5), 5 mM DTT («Euromedex»,
France), 0.1 mM ATP («Si gma», USA), 10 mM Mg-
Cl2 («Sigma», USA) for 1 h at 37 C with further ki-
nase inactivation during 20 min at 65 C.
Amplifi cation of the DTDTT gene was performed
as follows: denaturation – 1 min, 94 C; annealing –
1 min, 50 C; elongation – 1 min, 72 C (totally – 30
cycles) in 50 mM Tris-HCl (pH 9.0), 1.5 mM MgCl2,
20 mM ammonium sulfate («Merck», Germany),
1 μl of genomic DNA, 0.2 mM dNTP («Sigma»,
USA), 40 pmol of each primer and 2.5 units of Taq
DNA-polymerase («Stratagen», USA). The PCR fra g-
ment of about 460 bp was obtained and cloned fi rst
into pCRII-TOPO vector («Invitrogen», USA) using
Zero-Blunt-TOPO PCR cloning Kit («Life Technolo-
gies», USA). Top10 E.coli cells («Invitrogen», USA)
were used for the transformation. Screening for the
positive clones was performed by GeneJET Plasmid
Miniprep Kit («Thermo Scientifi c», Lithuania). The
positive clones were identifi ed by NcoI and HindIII
181
Cloning, expression and purifi cation of D-Tyr-tRNATyr-deacylase from Thermus thermophilus
restriction. The correct sequence of DTDTT ge ne
was confi rmed by DNA sequencing. Then the DTDTT
gene was excited by NcoI and Hind III Fast Digest
(«Thermo Scientifi c», Lithuania) restrictases and
ligated into pProEXHTb vector (EMBL, France),
previously dephosphorylated by 1 unit of shrimp pho s-
phatase in 1X reaction buffer («Roche», Switherland).
1 unit of T4-DNA-ligase in 1X ligase buffer («Ther-
mo Scientifi c», Lithuania), supplemented with 1mM
spermidine («Sigma», USA) and 1 mM ATP (for
20 h at 14 C) was used for ligation. Top10 E. coli
cells were transformed by ligation mixture using «Bio-
Rad» (USA) electroporation system.
Test of DTDTT expression in different media. E. co-
li BL21(DE3)pLysS cells were electroporated by pPro
ExHTb-DTD plasmid. Analysis of an expression le-
vel was performed in LB (Lauria-Broth) medium
(10 g/l tryptone, 5 g/l yeast extract («Difco», USA),
10/l g NaCl («Helicon», Russian Federation), TB
(Terrifi c-Broth) (12 g/l tryptone, 24 g/l yeast extract,
2.5 % glycerol, 2.31 g/l KH2PO4 and 12.54 g/l K2HPO4
(«Helicon», Russian Federation), P (phosphate-me-
dium) (10 g/l yeast extract, 0.4 % glucose («Heli-
con», Russian Federation), 5.6 g/l KH2PO4 and 28.9
g/l K2HPO4), 2x TY (16 g/l tryptone, 10 g/l yeast
extract, 5 g/l NaCl) supplemented with ampicilin
and chloramphenicol («Euromedex», France). Pre-
culture (5 ml) was grown overnight at 37 C. Culture
(50 ml of each medium) was inoculated with pre-
culture in dilution 1:100. Bacterial growth was con-
tinued to A600 = 0.6 and 0.6 mM IPTG («Thermo Sci-
entifi c», Lithuania) induction was performed for 5 h.
The extract of soluble proteins was prepared from 1
ml of culture volume according to the Zerbs et al.
protocol for the analysis of protein solubility [21].
DTDTT purifi cation by affi nity, anion exchange
and size-exclusion chromatography. Pre-culture of
E. coli BL21(DE3)pLysS cells (50 ml), carrying the
recombinant plasmids, was grown overnight at 37 C
in Terrifi c-Broth with appropriate antibiotics. The
cu lture (2.5 L) was inoculated with pre-culture in di-
lution 1:100 and grown at 37 C on TB medium sup-
plemented with ampicillin (100 μg/ml) and chloram-
phenicol (35 μg/ml). The expression of DTDTT was
induced by the addition of 0.6 mM IPTG at A600 = 0.6
and the culture growth continued for 4 h at 37 C.
The cells were harvested by the centrifugation for
15 min at 6000 × g (4 C). The bacterial cell pellet
was resuspended in 70 ml of 25mM Tris-HCl
(pH 7.5), 1mM PMSF, 10 mM β-mercaptoethanol
supplemented with 1.5 tablets of EDTA free protease
inhibitors cocktail. The cells were incubated on ice
for 30 min and then disrupted by sonication 8 × 30 sec
with 1 min breaks (4 C). All subsequent steps were
conducted at 4 C. The cell debris was precipitated
by centrifugation at 20 000 × g. The clear superna-
tant was recovered and concentrations of sodium
chloride and imidazole («Sigma», USA) were ad-
justed to 300 mM and 10 mM, respectively. The ob-
tained solution was mixed with Ni-NTA Sepharose
Fast Flow resin (5 ml of 50 % slurry, «GE He alth-
care», Sweden), pre-equilibrated with the same buff-
er, and incubated for 1.5 h on the rotor shaker at 130
rpm. The resin was washed with buffer A (25 mM
Tris-HCl, pH 7.5, 0.1 mM PMSF, 1 mM β-mer cap to-
ethanol, 300 mM NaCl, 10 mM imidazole) and then
with buffer A containing 600 mM NaCl. DTDTT
was eluted from the column by 400 mM imidazole in
buffer A. The collected fractions were analyzed by
SDS-PAGE. The purest fractions were combined and
dialyzed overnight against an appropriate buffer for
TEV protease digestion – buffer B (50 mM Tris-HCl
(pH 7.5), 0.1 mM PMSF, 1 mM DTT, 0.5 mM ED TA).
After the dialysis His-tag-residues were cut off from
DTDTT by recombinant TEV protease as follows:
1 A280 of TEV per 5 A280 of DTD during overnight
digestion at 4 °C [22]. The resulting solution from the
fi st purifi cation step was diluted to 1 A280 unites/ml
and applied on Q-Sepharose Fast Flow column
(«Pharmacia», Sweden) (1.35 x 4 cm, V = 6 ml), pre-
equilibrated by buffer B. A column was washed by the
same buffer. The elution was performed at a fl ow rate
of 0.6 ml/min with a linear gradient of 200-800 mM
NaCl (70 ml).
The protein-containing fractions were detected by
Bradford assay, the DTD-containing fractions we re
analyzed by 15 % SDS-PAGE. The collected frac tions
containing DTDTT were dialyzed overnight against
25 mM Tris-HCl (pH 7.5), 1 mM DTT at 4 C and
concentrated on 10 kDa Centricon («Merck», Ger-
182
M. Yu. Rybak, O. P. Kovalenko, I. A. Kryklyvyi, M. A. Tukalo
Western blot analysis of DTDTT
before and after TEV protease treatment
The proteins were separated in 15% SDS-PAGE and
transferred onto a prepared 0.45 μm polyvinyl dif-
luoride (PVDF) membrane (incubated for 1 min with
MeOH and rinsed once by Towbin buffer («Bio-
Rad», USA)) on Trans-Blot Semi-Dry electrophoret-
ic transfer system («Bio-Rad», USA). The mem-
brane was blocked overnight by 5 % non-fat milk in
PBST buffer solution (PBS plus 0.5 % Tween-20).
After blocking, membrane was incubated with mouse
anti-His mono- clonal antibodies («Sigma», USA) in
dilution 1 : 6000 for 1 h at room temperature. Then,
the membrane was extensively washed by PBST
buf fer (4 times × 5 min) and treated with secondary
anti-mouse antibodies (Jackson Immuno Research
Inc., USA), conjugated to peroxidase, at 1 : 10000
working dilution for 1 h. After this incubation the
extensive (4 times × 5 min) washing with PBST was
performed. The immune complexes were detected
by ECL detection kit (EMD Millipore Immobilon
Western Chemiluminescent HRP Substrate) («Mili-
pore», USA) using X-ray fi lm.
Results and Discussion
Creation of DTD expressing construction and expre-
ssion of recombinant protein in different media. Previ-
ously, we tried to express the DTD gene from T. ther-
mophilus in pET15b, pET28b and pET29b vectors
(under control of T7-promotor and lac-operator), but it
resulted in low expression level of the target protein
even after 24 h of IPTG induction. To overcome this
problem we decided to switch to pProEXHTb expres-
sion vector, which possess’ Trc promoter. pProEXHTb
was earlier shown to produce large quantities of the tar-
get proteins, during a short time IPTG induction [24].
The expression level of DTDTT in E. coli BL21
(DE3)pLysS cells was checked under varied IPTG con-
centrations and in several media (LB, TB, P, 2xTY).
The best conditions obtained were as follows: 4–5 h
of 0.6 mM IPTG induction at 37 C in Terrifi c Broth
medium (Fig. 1). These conditions were further used
for the preparative DTDTT expression.
many) at 5500 rpm to 4.54 A280 unites/ml (≈ 12 mg/
ml).
To separate our target protein from the high mo-
lecular weight contaminations we used a size-exclu-
sion chromatography on Hi-Load 16/60 Superdex
200 (150 ml, «Pharmacia Biotech», Sweden) co-lumn,
pre-equilibrated with 25 mM Tris-HCl (pH 7.5),
1 mM DTT, 150 mM NaCl, 0.003 % NaN3, with a
fl ow rate of 0.5 ml/min.
The eluted fractions were col lected and analyzed
by 15 % SDS-PAGE. The de acylase containing frac-
tions were combined and con centrated to 8 mg/ml.
The enzyme was supplemented by 50 % glycerol
and stored at –20 C.
The protein concentrations were determined by
the Bradford assay using Roti®-Quant («Roth», Ger-
many) [23]. Light absorption coeffi cient at 280 nm
(ε280 = 5960 M–1 cm–1) and absorbance of 0.1 % solu-
tion (A280 (1 mg/ml) = 0.354 unites · mg–1 · ml) were
calculated from the amino acid sequence of the DT-
DTT (ProtParam tool, ExPASy, Swiss Port) and used
for determination of the enzyme concentration.
Analytical gel fi ltration of proteins
To determine the approximate molecular weight of
DTDTT the gel fi ltration on Hi-Load 16/60 Super-
dex S 200 (150 ml, «Pharmacia Biotech») was
used. The column was pre-equilibrated with 25 mM
Tris-HCl (pH 7.5), 1 mM DTT, 150 mM NaCl,
0.003 % NaN3. All samples were run at 1 ml/min
fl ow rate. The void column volume (Vo) was deter-
mined by blue dextran (2 MDa). A set of proteins
were used for the column calibration: ferritin
(450 kDa), catalase (240 kDa), β-amilase (200kDa),
alcohol dehydrogenase (150 kDa), bovine serum
albumin (66 kDa), ovalbumin (45 kDa), carboan-
hidrase (29 kDa), cytochrome c (12,4 kDa).
The molecular weight of DTDTT was determined
by a comparison of its Ve / Vo index with those of
the known protein standards. The logarithms of the
molecular weights of marker proteins were plotted
against their appropriate ratios of the elution vol-
ume to the column void volume (Ve / Vo). Calibra-
tion curve is shown in Fig. 6.
183
Cloning, expression and purifi cation of D-Tyr-tRNATyr-deacylase from Thermus thermophilus
Fig. 2. Affi nity purifi cation of His-DTDTT on Ni-NTA column.
15 % SDS-PAGE of fractions: M – protein marker (Roti-Mark
10–150); 1 – soluble protein lysate; 2 – fl ow-through fraction
after Ni-NTA column; 3 – wash Ni-NTA column; 4 – combined
eluted DTDTT fraction; 5 – His-DTDTT after dialysis. The po-
sition of His-DTDTT on the gel is indicated by arrow
Fig. 3. 15 % SDS-PAGE (left panel) and Western blot (right pa-
nel) analysis of His-DTDTT before (1) and after (2) TEV pro-
tease treatment
Purifi cation of His-DTDTT
The fi rst step of the His-DTDTT purifi cation was the
affi nity chromatography on Ni-NTA. The result is
presented in Fig. 2. The enzyme, eluted from the col-
umn by 400 mM imidazole, contained the contami-
nations of higher molecular weight proteins. Wash-
ing the column with a buffer supplemented with
20 mM imidazole slightly increased the DTDTT pu-
rity but decreased its yield. In addition, washing the
column with 1 M NaCl did not signifi cantly dimin-
ish the amount of impurities (data not shown). Un-
fortunately, we could not improve the quality of the
DTDTT preparation after this step of purifi cation.
Fig. 4. 15 % SDS-PAGE of fractions after Q-Sepharose chromatography: M – protein marker (RPN 58100, «Amersham»); A – pro-
tein, loaded onto the column; lanes 1–11 – fractions obtained during the gradient elution from Q-Sepharose column. The position of
DTDTT on the gel is indicated by arrow
Fig. 1. DTDTT expression in TB medium. 15 % SDS-PAGE of the
soluble protein extracts from BL21(DE3)pLysS: M – protein mark-
er (SDS-PAGE Standards, Low 6-200 «Bio-Rad»); 1 – extract from
non-induced bacteria; 2–6 – extracts after 1–5 hours of 0.6 mM
IPTG induction. The position of His-DTDTT is indicated by arrow
184
M. Yu. Rybak, O. P. Kovalenko, I. A. Kryklyvyi, M. A. Tukalo
Fig. 6. Determination of DTDTT molecular weight by size-ex-
clusion chromatography on Superdex S 200. Calibration of Su-
perdex S 200 column was performed as described in «Materials
and methods» (Fer-di – ferritin dimer; Fer-mono – ferritin mon-
omer; β-A – β-amilase; Cat – catalase; AcDeh – alcohol dehy-
drogenase; BSA – bovine serum albumin; Ova – ovalbumin;
DTDTT – D-Tyr-tRNATyr-deacylase; CA – carboanhidrase; Cyt.c –
cytochrome c). The position of DTDTT is indicated by a circle
Fig. 5. Purifi cation of DTDTT by size-exclusion chromatography: A – elution profi le of DTDTT on Superdex S 200 column; B – 15 %
SDS-PAGE of fractions after Superdex 200: M – protein marker (Roti-Mark 10–150); 1 – DTDTT applied onto Superdex 200 column;
2 – empty lane; lanes 3–8 – corresponding fractions obtained after the gel fi ltration. The position of DTDTT is indicated by arrow
BA
To remove His-tag from the His-DTDTT enzyme
we used the recombinant TEV protease as descri-
bed in «Materials and methods». The effi ciency of
cleavage was checked by SDS-PAGE and Western
blot analysis with anti-His antibodies (Fig. 3). The
absence of a signal on the lane 2 of Western blot
panel confi rms complete His-tag removing from
DTDTT. Moreover, there is a shift of the DTDTT
migration on SDS-PAGE (lane 2, left panel) after
the TEV protease treatment. Thus, the His-tag cle a-
vage from His-DTDTT can be simply monitored by
SDS-PAGE.
It is worth noting that the ratio A260/A280 of DT-
DTT after the affi nity purifi cation step and His-tag
cleava-ge was about 1.0 that may refl ect the pres-
ence of nuc- leic acid contaminations. To remove
the nucleic acid fragments we applied an anion-
exchange chromatography. Unfortunately, during
this step we could not get rid of the protein con-
tamination present in the DTDTT preparation. We
applied various linear gradients (from 0 to 1M NaCl
and from 50 mM to 800 mM NaCl), but this did not
improve the quality of DTDTT. Finally, we used
the gradient from 200 to 800 mM of NaCl, which
allowed us to remove the nucleic acids and some
protein contaminations. After Q-Sepharose column,
DTDTT had typical absorbance ratio A260/A280 =
0.5–0.6. SDS-PAGE of fractions obtained after Q-
Sepharose is presented in Fig. 4.
To get rid of the higher molecular weight impurities
in the DTDTT preparation we performed a size-exclu-
sion chromatography as a fi nal step of the purifi cation
procedure. The elution profi le of DTDTT from the col-
umn is shown in Fig. 5, B. As can be judged from the
elution profi le, DTDTT was effi ciently separated from
the contaminating proteins and eluted from the column
as a single peak (Fig. 5, A). According to SDS-PAGE
185
Cloning, expression and purifi cation of D-Tyr-tRNATyr-deacylase from Thermus thermophilus
(Fig. 5, B) the purity of DTDTT may be more than 90 %.
Molecular weight determination
of D-Tyr-tRNATyr-deacylase
The molecular weight of the D-Tyr-tRNATyr deacy-
lase was deduced from a comparison of its elution
time on Superdex S 200 column with the proteins of
known molecular weight. Column was calibrated as
described in «Materials and methods». The elution
volume (Ve) of DTDTT from Superdex S 200 was de-
termined to be 11 ml. Ve / Vo index of DTDTT was cal-
culated to be 1,888. According to the calibration curve
(Fig. 6) the molecular weight of DTDTT was estimate
to be 32 kDa. Based on the amino acid sequence of
DTDTT (152 a. a. residues), its theoretical molecular
weight is 16.7 kDa. Thus, the purifi ed recombinant
DTDTT most probably is a dimer in solution. How-
ever, a monomeric unglobular form of this protein
with an elongated shape could not be excluded.
Conclusions
The cDNA encoding D-Tyr-tRNATyr-deacylase from
T. thermophilus was cloned into pProEXHTb vector
and successfully expressed in BL21(DE3)pLysS stra-
in in TB medium. The purifi cation procedure descri-
bed here allows obtaining 2 mg of the pure enzyme
from 1 L of the bacterial culture. According to the gel
fi ltration analysis recombinant DTDTT may exist as a
dimer in solution. The obtained protein will be used
for further structural and functional studies.
Acknowledgements
We are grateful to Dr. O. Gudzera for TEV protease
purifi cation, Dr. O. Malanchuk for the help with
Western blot analysis and Dr. V. Shalak for helpful
discussion and advice in manuscript preparation.
REFERENCES
1. Corrigan JJ. D-amino acids in animals. Science. 1969;164
(3876):142–9.
2. Leiman SA, May JM, Lebar MD, Kahne D, Kolter R, Losick R.
D-amino acids indirectly inhibit biofi lm formation in Bacil-
lus subtilis by interfering with protein synthesis. J Bacteriol.
2013;195(23):5391–5.
3. Fujii N. D-amino acid in elderly tissues. Biol Pharm Bull.
2005;28(9):1585–9.
4. Liu W, Liu C, Zhu JX, Li AH, Zhao ZQ, Yin B, Peng XZ. D-
Tyr-tRNA(Tyr) deacylase, a new role in Alzheimer’s-asso-
ciated disease in SAMP8 mice. Chin Med Sci J. 2010;25
(2):90–4.
5. Friedman M, Gumbmann MR. The nutritive value and safe-
ty of D-phenylalanine and D-tyrosine in mice. J Nutr. 1984;
114(11):2089–96.
6. Friedman M. Formation, nutritional value, and safety of D-
amino acids. Adv Exp Med Biol. 1991;289:447–81.
7. Friedman M. Chemistry, nutrition, and microbiology of D-
amino acids. J Agric Food Chem. 1999;47(9):3457–79.
8. Friedman M. Origin, microbiology, nutrition, and pharmaco-
lo gy of D-amino acids. Chem Biodivers. 2010;7(6):1491–530.
9. Calendar R, Berg P. D-Tyrosyl RNA: formation, hydrolysis
and utilization for protein synthesis. J Mol Biol. 1967;26(1):
39–54.
10. Soutourina J, Plateau P, Blanquet S. Metabolism of D-ami-
noacyl-tRNAs in Escherichia coli and Saccharomyces cer-
evisiae cells. J Biol Chem. 2000;275(42):32535–42.
11. Wydau S, van der Rest G, Aubard C, Plateau P, Blanquet S.
Widespread distribution of cell defense against D-aminoa-
cyl-tRNAs. J Biol Chem. 2009;284(21):14096–104.
12. Soutourina J, Plateau P, Delort F, Peirotes A, Blanquet S. Fun-
ctional characterization of the D-Tyr-tRNATyr deacylase from
Escherichia coli. J Biol Chem. 1999;274(27):19109–14.
13. Soutourina J, Blanquet S, Plateau P. D-tyrosyl-tRNA(Tyr)
metabolism in Saccharomyces cerevisiae. J Biol Chem. 2000;
275(16):11626–30.
14. Wydau S, Ferri-Fioni ML, Blanquet S, Plateau P. GEK1, a
gene product of Arabidopsis thaliana involved in ethanol
tolerance, is a D-aminoacyl-tRNA deacylase. Nucleic Acids
Res. 2007;35(3):930–8.
15. Zheng G, Liu W, Gong Y, Yang H, Yin B, Zhu J, Xie Y, Peng X,
Qiang B, Yuan J. Human D-Tyr-tRNA(Tyr) deacylase con-
tributes to the resistance of the cell to D-amino acids. Bio-
chem J. 2009;417(1):85–94.
16. Ferri-Fioni ML, Schmitt E, Soutourina J, Plateau P, Mechu-
lam Y, Blanquet S. Structure of crystalline D-Tyr-tRNA(Tyr)
deacylase. A representative of a new class of tRNA-depend-
ent hydrolases. J Biol Chem. 2001;276(50):47285–90.
17. Lim K, Tempczyk A, Bonander N, Toedt J, Howard A, Eisen-
stein E, Herzberg O. A catalytic mechanism for D-Tyr-tRNA
Tyr deacylase based on the crystal structure of Hemophilus
infl uenzae HI0670. J Biol Chem. 2003;278(15):13496–502.
18. Ferri-Fioni ML, Fromant M, Bouin AP, Aubard C, Lazennec C,
Plateau P, Blanquet S. Identifi cation in archaea of a novel
D-Tyr-tRNATyr deacylase. J Biol Chem. 2006;281(37):
27575–85.
19. Ahmad S, Routh SB, Kamarthapu V, Chalissery J, Muthuku-
mar S, Hussain T, Kruparani SP, Deshmukh MV, Sankara-
narayanan R. Mechanism of chiral proofreading during
translation of the genetic code. Elife. 2013;2:e01519.
20. Marmur J. A procedure for the isolation of deoxyribonu-
cleic acid from micro-organisms. J Mol Biol. 1961; 3(2):
186
M. Yu. Rybak, O. P. Kovalenko, I. A. Kryklyvyi, M. A. Tukalo
208–IN1.
21. Zerbs S, Frank AM, Collart FR. Bacterial systems for pro-
duction of heterologous proteins. Methods Enzymol. 2009;
463:149–68.
22. Waugh DS. An overview of enzymatic reagents for the remo-
val of affi nity tags. Protein Expr Purif. 2011;80(2):283–93.
23. Bradford MM. A rapid and sensitive method for the quanti-
tation of microgram quantities of protein utilizing the princi-
ple of protein-dye binding. Anal Biochem. 1976;72:248–54.
24. Polayes D. Prokaryotic protein expression and purifi cation
with the ProEXTM HT expression system. Focus. 1996; 18(2):
50–3.
Клонування, експресія та очистка
D-Тир-тРНКТир-деацилази Thermus thermophilus
М. Ю. Рибак, О. П. Коваленко, І. А. Крикливий, М. А. Тукало
D-Тир-тРНКТир-деацилаза (DTD) є консервативним білком,
що забезпечує додатковий корегувальний етап гідролізу по-
милково аміноацильованих субстратів D-Тир-тРНКТир DTD
прискорює гідроліз ефірного зв’язку в комплексі D-Тир-
тРНКТир, утворюючи вільну тРНК та D-Тир, таким чином
попереджуючи включення D-амінокислот до білків. Деаци-
лаза розрізняє D- та L-аміноацильні залишки в аміноаци-
льованій тРНК та не гідролізує останні. Структурні основи
такої специфічності та механізм гідролізу D-аміноацил-тРНК
за участі DTD є недостатньо зрозумілими. Мета. Клону-
вати D-Тир-тРНКТир-деацилазу з T. the rmophilus (DTDTT),
оптимізувати умовт її експресії в E. coli, розробити ефектив-
ний метод очищення з високим виходом високоякісного
ферменту для структурно-функціональних досліджень. Ме-
тоди. Ампліфікацію та клонування гена DTD з геномної
ДНК T. thermophilus в pProEXHTb експресуючий вектор
проведено стандартними молекулярно-біологічними мето-
дами. Очистку рекомбінантного DTD білка проводено хро-
матографічно. Залишки гістидину відщеплено від DTD про-
теазою вірусу тютюну (TEV), ефективність реакції переві-
рено Вестерн-блот аналізом з анти-His-антитілами. Моле-
кулярну масу очищеної DTDTT визначено гель-фільтрацією.
Результати. Отримана конструкція pProEXHTb, що містить
DTD послідовність з T. thermophilus, експресовано в E.coli
штаму BL21(DE3)pLysS. Цільовий рекомбінантний білок
виділено і очищено комбінацією хроматографій: афінної
(Ni-NTA), аніон-об мін ної (Q-Sepharose) та гель-фільтрації
(Superdex S 200) до чистоти 90%. Розроблений метод дозво-
ляє отримати до 2 мг цільового білка з 1 л бактеріальної куль-
тури. Визначена молекулярна маса очищеної DTD з T.
thermophilus – 32 кДа свідчить на користь її димерної струк-
тури. Висновки. Отриманий вектор pProEXHTb, що містить
DTD з T. thermophilus і підібрані умови очистки дозволяють
отримати рекомбінантний DTD в кількостях достатніх для
подальших структурно-функціональних досліджень.
Ключов і слова: D-амінокислоти, D-Тир-тРНКТир-деа ци-
лаза Thermus thermophilus, клонування, експресія, очистка.
Клонирование, экспрессия и очистка
D-Тир-тРНКТир-деацилазы Thermus thermophilus
М. Ю. Рыбак, О. П. Коваленко, И. А. Крикливый, М. А. Тукало
D-Тир-тРНКТир-деацилаза (DTD) является консервативным
белком, обеспечивающим дополнительный корректирую-
щий этап гидролиза ошибочно аминоацилированных суб-
стратов D-Тир-тРНКТир. DTD ускоряет гидролиз эфирной
связи в комплексе D-Тир-тРНКТир, предупреждая, таким об-
разом, ошибочное включение D-аминокислот в белки. Деа-
цилаза различает D- и L-аминоацильные остатки в аминоа-
цилированной тРНК и не гидролизирует последние. Струк-
турные основы такой спе цифичности и механизм гидролиза
D-аминоацил-тРНК при участии DTD являются недостаточ-
но изученными. Цель. Клонировать D-Тир-тРНКТир-деаци-
лазу из T. ther mo phi lus (DTDTT), оптимизировать условия
её экспрессии в E. coli и разработать эффективный метод
очистки с высоким выходом высококачественного фермента
для дальнейших структурно-функциональных исследова-
ний. Методы. Для амплификации и клонирования гена DTD
из геномной ДНК T. thermophilus в pProEXHTb экспресиру-
ющий вектор использованы стандартные молекулярно-био-
логические методы. Очистку рекомбинантного DTD белка
проводили хроматографически. Остатки гистидина отщепи-
ли протеазой вируса табака (TEV), эффективность реакции
проверили Вестерн-блот анализом с анти-His-антителами.
Молекулярный вес очищенной DTDTT определили гель-
фильтрацией. Результаты. Полученная конструкция
pProEXHTb, содержащая DTD последовательность T. ther-
mophilus, экспрессирована в клетках E. coli штамма BL21(DE3)
pLysS. Последовательными хроматографиями: афин ной (Ni-
NTA), анион-обменной (Q-Sepharose) и гель-фильтрации
(Superdex S 200) получен гомогенный белок с чистотой более
90 %. Разработанная методика позволяет получить до 2 мг
целевого белка / 1 л бактериальной культуры. Определенный
молекулярный вес очищенной DTD из T. ther mophilus – 32
кДа свидетельствует о ее димерной структу ре. Выводы. По-
лученная конструкция pProEXHTb, содержаащя DTD после-
довательность T. thermophilus и подобранные условия очист-
ки позволяют получить рекомбинантный DTD для дальней-
ших структурно-функциональных исследований.
Ключевые слова: D-аминокислоты, D-Тир-тРНКТир-деа ци-
лаза Thermus thermophilus, клонирование, экспрессия, очистка.
Received 30.03.2015
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