Cloning, Purification, and Polymerization of Capsicum annuum

Recombinant � and � Tubulin

Myung-Hyun JANG,1 Jungmok KIM,1 Satish KALME,1 Jin-Wook HAN,1 Han-Sang YOO,2

Jinheung KIM,3 Bon-sung KOO,4 Sung-Kun KIM,5 and Moon-Young YOON1;y

1Department of Chemistry, Hanyang University, Seoul 133-791, Korea2Department of Infectious Diseases, College of Veterinary Medicine,

BK21 for Veterinary Science and KRF Zoonotic Disease Priority Research Institute,

Seoul National University, Seoul 151-741, Korea3Department of Chemistry, Ewha Women’s University, Seoul 120-750, Korea4Microbial Function Team, National Institute of Agricultural Biotechnology, RDA, Suwon 441-707, Korea5Department of Chemistry and Biochemistry, Baylor University, Waco, Texas 76798-7348, USA

Received December 6, 2007; Accepted December 25, 2007; Online Publication, April 7, 2008

[doi:10.1271/bbb.70794]

� and � tubulin genes were cloned from the Capsicum

annuum leaves using rapid amplification of cDNA ends

(RACE)-PCR. Nucleotide sequence analysis revealed

that 1,353 bp Capsicum annuum �=�-tubulin (CAnm

�=�-TUB) encodes a protein of 450 amino acids (aa)

each. The recombinant �=� tubulin was overexpressed

mainly as an inclusion body in Escherichia coli BL21

(DE3), upon induction with 0.2mM isopropyl-�-D-thio-

galactopyranoside (IPTG), and its content was as high

as 50% of the total protein content. Effective fusion

protein purification and refolding are described. The

average yields of � and � tubulin were 2.0 and 1.3mg/l

of culture respectively. The apparent molecular weight

of each tubulin was estimated to be 55 kDa by SDS-

polyacrylamide gel electrophoresis (PAGE). The tubulin

monomers were found to be assembly competent using a

standard dimerization assay, and also retained antige-

nicity with anti-His/T7 antibodies. The purified tubulins

were polymerized to microtubule-like structures in the

presence of 2mM guanosine 50-triphosphate (GTP).

Key words: Capsicum annuum; dimerization; microtu-

bule; pepper; rapid amplification of cDNA

ends (RACE)-PCR

The basic building block of microtubules is tubulin,a heterodimeric protein consisting of a non-covalentinteraction of two related approximately 50 kDa poly-peptides known as �- and �-tubulin. Tubulin dimerspolymerize in a head-to-tail manner to form 4 to 5 nmlinear protofilaments, typically 13 of which are laterallyassociated in the wall of the 24-nm diameter micro-tubule.1) During the plant cell cycle, microtubules are

reversibly polymerized to form functionally distinctarrays that mediate sub-cellular motility phenomenaand provide cytosolic organization. They are involvedin many essential functions in plant cells, includingcellular morphogenesis, organelle and vesicle transport,chromosome segregation, cell wall formation, and celldivision.1,2)

Phytophthora blight is caused by the oomycetepathogen P. capsici. It is a devastating disease of pepper(C. annuum) in South Korea and worldwide. Micro-tubules are a known target of fungicides such as thebenzimidazoles. Since some fungicides and herbicidesdisplay unacceptable phytotoxicity,3,4) it is importantthat any antimitotic compound for plant pathogenicfungi should be non-phytotoxic.Several methods have been developed to isolate

native tubulin from plant material,5–7) but lower sol-ubility is a major concern in large-scale applications.The purpose of this study was to overcome thedifficulties in preparing sufficient quantities of planttubulin by purification of recombinant � and � tubulin ofC. annuum (CAnm �-TUB/�-TUB). Dimerization oftubulin and the formation of microtubule-like structureswere studied. The present work provides information ontubulin of the Solanaceae family, promoting structuraland functional research into plant tubulin. In future, theC. annuum microtubule will be used as a control inscreening of antimitotic compounds against P. capsici.

Materials and Methods

Plant material, strain, plasmid, and enzymes. C. ann-uum fresh leaves were obtained from a local farm

y To whom correspondence should be addressed. Tel: +82-2-2220-0946; Fax: +82-2-2299-0762; E-mail: myyoon@hanyang.ac.kr

Abbreviations: aa, amino acids; CAnm �-TUB, Capsicum annuum �-tubulin; CAnm �-TUB, Capsicum annuum �-tubulin; GTP, guanosine 50-

triphosphate; IPTG, isopropyl-�-D-thiogalactopyranoside; PAGE, polyacrylamide gel electrophoresis; RACE, rapid amplification of cDNA ends

Biosci. Biotechnol. Biochem., 72 (4), 1048–1055, 2008

(Seoul, South Korea). E. coli BL21 (DE3) (Novagen,Madison, WI) and plasmid pET28a (Novagen) wereused in gene expression. Restriction and modifyingenzymes were purchased from Takara Bio (Kyoto,Japan), and used according to the recommendations ofthe supplier. Other DNA manipulation experimentswere performed as described by Sambrook et al.8)

cDNA synthesis, partial gene of �=� tubulin, andrapid amplification of cDNA ends. To isolate C. annuumcDNA, the RT-PCR and rapid amplification of cDNAends (RACE) methods were followed. For this, totalRNA was isolated from 100mg of C. annuum freshleaves (ground in liquid nitrogen) using an Easy blue�Kit (iNtRON Biotechnology, Seoul, South Korea), andreverse transcribed using Takara RNA PCR kit (AMV),Ver.3.0. The second strand of cDNA was synthesizedusing oligo(dT)17 adaptator primer: M13 primer M4 (50-GTTTTCCCAGTCACGACTTTTTTTTTTTTTTTTT-30). Degenerate primers, 50-ATGAGGGAGTGCATTT-CAATCCAC and 30-ACCTCAGCAACACTTGTT-GAGTTG, were designed on the basis of an evolutio-narily conserved region of tubulin coding sequences,and used to obtain a partial gene of C. annuum �tubulin. In isolating partial � tubulin gene, the primerswere 50-AACATATGATGAGAGAAATCCTTCACA-TTCAAGGAGG and 30-AACTCGAGTTAGGATTCA-TATTGATGATCATCAGCGGC, containing NdeI andXhoI restriction sites (underlined) respectively.

To isolate the full-length cDNA, 50 and 30 RACE wasperformed using gene-specific primers designed on thepartial cDNA sequences obtained. In the 50 RACEmethod, the specific reverse primer (for � tubulin, 50-TGGCTGCATCTTCTTTGC-30 and for � tubulin, 50-CGAACAACATCTAGAACAG-30) was used in reversetranscription with terminal deoxynucleotidyl transferase.The first cDNA strand obtained was poly-(dA)-tailed,and it was amplified with an oligo(dT) adaptator primer(M13 primer M4) and the specific primer for � (50-ACTTCATCAATGACAGTGG-30) or � (50-TCCAAA-GACAAAGT AATCAGGAC-30) tubulin. To isolate the30 end of the cDNA, the 30 RACE protocol was initiatedwith synthesis of the first cDNA strand using theoligo(dT)17 adaptator primer. This first strand was thenamplified with the specific primer (for � tubulin, 50-AGTGTGGTATCAACTATCAGC-30, and for � tubu-lin, 50-CAATGTCAAGTCTAGCGT GTG-30) to gener-ate a short second strand, ending in a sequencecomplementary to the original adaptator sequence.Thereafter, PCR was initiated using the specific primerand an adaptator primer.

Cloning of full length � and � tubulins. The 50 and 30

RACE products were used to generate primers corre-sponding to the beginning and the end of the codingregion. For � tubulin, the 50-full primer was 50-AAGTCGACAAATGAGAGAATGCATTTCTATTCA-CATTGG-30 and the 30-full primer was 50-AA-

GCGGCCGCTTAGTATTCCTCATGATCATCAT-30.These primers contained restriction sites for SalI andNotI (underlined). For � tubulin, the 50-full primerwas 50-AACATATGATGAGAGAAATCCTTCACA-TTCAAGGAGG-30 and the 30-full primer was 50-AACTCGAGTTAGGATTCATATTGATCATCAGGG-GCCT-30. These primers contained NdeI and XhoIrestriction sites (underlined). The total RNA isolatedas described above was reverse transcribed using thesetwo primers. The sequencing of both the strands wasaccomplished using BigDyeTM terminator. The reactionproduct was purified using ethanol precipitation, andsequenced with an automatic sequencer 3730xl (Macro-gen, Seoul, South Korea). The � and � tubulin insertswere then cloned into the SalI/NotI and NdeI/XhoI sitesof pET28a (Novagen), respectively, producing fusionproteins with N-terminal hexahistidine tags.

Culture and isolation of inclusion bodies. Theexpression plasmids were transformed into E. coliBL21 (DE3) by the heat shock method. The transformedbacteria were grown at 37 �C in LB medium containing100 mg/ml kanamycin until the A600 of the culturereached 0.5. Isopropyl-�-D-thiogalactopyranoside (0.2mM, IPTG) was added to induce expression of recombi-nant �=� tubulin at 18 �C overnight. The culture grownwas collected by centrifugation and suspended in 40ml20mM Tris buffer, pH 8.0. The cells were disruptedby ultrasonication (Heat Systems, Boston, MA) using 60cycles, each of 1 s, at 15% output power. Cell-freeextract was centrifuged at 23;700� g for 10min at 4 �C,and the pellet was resupended in 40ml of isolationbuffer (20mM Tris, 300mM NaCl, and 2% Triton X-100,pH 8.0) for sonication, as described above. The pelletwas resuspended in isolation buffer and sonication wasrepeated twice.

Solubilization of inclusion bodies, refolding, andpurification. The pellet obtained after sonication wassuspended in 40ml of binding buffer (6 M guanidinehydrochloride, 20mM Tris, 300mM NaCl, 5mM imid-azole, and 1mM �-ME, pH 8.0) and incubated at 25 �Cwith gentle stirring for 30min. The supernatant wascollected by centrifugation at 23;700� g for 10min at4 �C. Undissolved particles were removed by filtrationthrough a 0.45-mm syringe filter (Millipore, Boston,MA).Supernatant containing � or � tubulin was loaded onto

a Ni2þ charged sepharose column pre-equilibrated withthe binding buffer (flow rate, 0.5ml/min). The columnwas washed with binding buffer (3 bed volumes), andthen with the washing buffer (6 M urea, 20mM Tris,300mM NaCl, 20mM imidazole, and 1mM �-ME,pH 8.0). The bound protein was refolded on-columnusing a decreasing gradient of urea (6-0 M), starting withthe washing buffer and finishing with the refolding buffer(20mM Tris, 300mM NaCl, 20mM imidazole, and 1mM

�-ME, pH 8.0). After completion of the gradient,washing was continued with two bed volumes of

Cloning, Purification, and Polymerization of C. annuum Tubulin 1049

refolding buffer. The refolded protein was eluted usingelution buffer (20mM Tris, 300mM NaCl, 0.5 M imid-azole, and 1mM �-ME, pH 8.0) with a linear gradientfrom 0.02 to 0.5 M imidazole. Fractions containing thetarget protein were dialyzed against 50mM Tris (pH 8.0),and concentrated by ultrafiltration using YM10 cut-offmembrane (Amicon, Boston, MA). The purity of � and �tubulin was assessed by SDS–PAGE9) resolved on a0.75mm 4% stacking/12% resolving minigel (Hoefer�,Amersham Biosciences, Boston, MA). Proteins werevisualized with Coomassie brilliant blue R-250 stain,and sizes were estimated from broad range precisionstandards (Sigma Aldrich, ST. Louis, Missouri).

Reconstitution of recombinant � and � tubulin inheterodimers. The His tag of alpha tubulin was removedby thrombin action (10U/mg protein) prior use indimerization reaction. Recombinant � (T7þHis�) and �(Hisþ) tubulin monomers (100 mg/ml) were dimerizedon Niþ2 charged sepharose resin in buffer containing0.1 M Hepes (pH 7.4), 1mM EDTA, and 1mM MgCl2,with or without 2mM GTP, at 25 �C for 20min (Fig. 1).The washing buffer contained 0.1 M Hepes (pH 7.4),50mM imidazole, 1mM MgCl2, and 1mM EDTA. Thedimerized tubulin was eluted with elution buffercontaining 1M imidazole (the other components weresame as in the washing buffer). The inclusion of � and �tubulin components in the dimer was analyzed bywestern blotting10) using primary monoclonal anti-his(Santa Cruz Biotechnology, Santa Cruz, CA) and anti-T7 (Chemicon International, Santa Cruz, CA) antibod-ies. Blotted bands were visualized using goat-anti-rabitIgG antibodies as secondary antibodies.

Microtubule formation assay. The time course ofmicrotubule assembly was monitored at A350

11) using anOptizen 2120 UV spectrophotometer equipped with adigital temperature control assembly. The reactionmixture (400 ml) contained 0.5mg/ml of � and �-tubulin

in polymerization buffer (80mM Pipes, pH 6.8, 1mM

EGTA, 1mM MgCl2, and 5% v/v glycerol). Thereaction was initiated by adding GTP at a finalconcentration of 2 or 5mM. The contents were mixed,and turbidity was measured at 37 �C over a time scaleof 95min, reading at every 5 s.

Transmission electron microscopy. Completely poly-merized tubulin mixtures were prepared for transmissionelectron microscopy (TEM).12) Briefly, 30 ml of poly-merized sample was immediately diluted with 10 ml of0.4% glutaraldehyde for 1min at room temperature.Tubulin solution (10 ml) was applied to 200-mesh,copper/formvar coated grids (EMS) for 1min, washedwith two drops of dH2O, and stained for 2min with adrop of 2% uranyl acetate. Excess stain was removed byblotting with filter paper. The samples were air dried andlater viewed using a transmission electron microscope(JEM 2010, JEOL, Tokyo).

Results and Discussion

Cloning and sequencing of the �=� tubulin geneCAnm �=� tubulin cDNAs were isolated by the

RT-PCR and RACE methods. Primers corresponding tothe 50- and 30- ends of the coding region of C. annuum �and � tubulin yielded a product of 1,353 bp (data notshown). The tubulin insert cloned into the pET28abacterial expression vector provided an N-terminalHis-tagged protein, which was purified in one step.The � and � tubulin gene sequences were 1,353 bp long,with Gþ C contents of 47 and 45% respectively, andwere predicted to encode a putative 450 aa proteinindividually (data not shown). These results were ingood agreement with the aa sequences reported for planttubulins, including Arabidopsis thaliana,13,14) Glycinemax L.,15) and Eleusine indica.16,17) At the nucleotidelevel, the CAnm �- and �-TUB genes shared 78–95and 76–93% homology with coding regions of planttubulin cDNAs.18) The genebank accession numbersfor the sequences reported in C. annuum are EF495257(� tubulin) and EF495259 (� tubulin).

Analysis of � and � tubulin sequencesThe predicted CAnm �-TUB aa sequence was

compared with all isotype tubulin sequences present inthe databases.18) The greatest similarity was found withthe �-tubulin family. In particular, the highest matchwas found with Nicotiana tabacum tubulin (99%identity). � tubulin from other plant sources, such asZea mays (tubulin � 3-chain), Brassica napus, Oryzasativa, and Eleusine indica, had 92–97% sequenceidentity with CAnm �-TUB (data not shown). Clustal Walignment19) of the deduced aa sequence of CAnm�-TUB with some �-tubulin sequences derived fromorganisms at different evolutionary levels exhibited highdegrees of homology (Fig. 2). The aa sequence ofCAnm �-TUB had 96, 88, 84, and 78% identity to

Washingα α + GTP

Washing

Add

Add

Ni+2 chargedsepharose

reaction –α + β

reaction –α + β + GTP

Detection using anti-T7-tag/His-tag antibody1

Elution

3 5

2 4

α β β

Control –only α

Fig. 1. Dimerization of Recombinant � and � Tubulin Monomers.

Numbers 1–5, fractions collected during washing or elution of the

sample.

1050 M.-H. JANG et al.

A. thaliana, Plasmodium falciparum, Drosophila mela-nogaster/Xenopus laevis, and Homo sapiens �-tubulinsrespectively. Although the carboxyl termini of �tubulins are highly divergent, a carboxyl terminaltyrosine is conserved. CAnm �-TUB showed stronghomology to � tubulins from animals and protists. This

homology indicates that the structural tubulin genepredates the plant-animal evolutionary split.Figure 2 shows the blocks of highly homologous

residues. The extremely homologous regions includedthe diagnostic portion from aa 401 to 409, which isconserved exclusively within the �-tubulin subclass.20)

Fig. 2. Homology between C. annuum and Other �-Tubulins.

Identical and conserved amino acids are denoted by � and :/., respectively. The amino acids highlighted in black/gray are discussed in the

text. DROME, D. melanogaster tubulin �-3 chain (P06605); XENLA, X. laevis (P08537); HUMAN, Human alpha 8-tubulin (Q9NY65);

ARATH, A. thaliana (P29511); PLAFK, P. falciparum (P14642); CAnm, C. annuum (ABP65320).

Cloning, Purification, and Polymerization of C. annuum Tubulin 1051

The potential GTP-binding site in CAnm �-TUB wasdeduced by conserved domain search,21) and locatedat residues 140 to 146. The cell attachment sequence,RGD tripeptide, was present at 320 to 322 residues. Thecharacteristic �-tubulin MRECI domain, which has beenhypothesized to be involved in autoregulated post-transcriptional mechanisms,22,23) was conserved inCAnm �-TUB at the N-terminal. The lysine residueat position 40, modified in many isotypes of �-tubulinby the addition of an acetyl group, was conserved.24,25)

The C-terminal domain of the tubulin family, whichplays a crucial role in tubulin polymerization and ligandinteraction, changes greatly among different organismsand among various isotypes. In CAnm �-TUB, thecarboxyl terminus region was not fully conserved fromthose of other �-tubulins. The tyrosine residue located atthe C-terminal end was conserved, indicating that thistubulin isotype is modified by the tyrosination/detyr-osination cycle, to which �-tubulin is generally sub-jected. Moreover, a specific C-terminal glutamateresidue, the usual substrate for polyglycylation andpolyglutamylation modification,26) was highly con-served, suggesting that it is an efficient substrate forthese post-translational modifications.

The BLAST of the CAnm �-TUB aa sequenceresulted in 100% sequence identity with Lycopersiconesculentum, and 93–99% with other plant � tubulins,viz., Zea mays (beta-1 chain), E. indica, A. thaliana,Eucalyptus grandis, Solanum tuberosum (beta-2 chain),and N. attenuata. Microtubule-dependent processes inplants and algae are very sensitive to low concentrationsof dinitroaniline and phosphoric amide herbicides ascompared to these processes in animals.27,28) Hencestructural comparisons of plant and animal tubulinsshould give insight into the relations among diversegroups and into distinct functional properties in thesetwo tubulin groups. Sequence alignment of the deducedaa sequences of the � tubulin cDNAs from differentgroups is shown in Fig. 3. The mutation sites, such asHis6, Glu198, Tyr50, and Arg241 for benzimidazoleresistance in fungi and plants,17,29,30) are highly con-served in CAnm �-TUB (Fig. 3). Ala165, a benzimida-zole-sensitive site in A. nidulans, is replaced by Leu168in CAnm �-TUB, similarly observed in E. indica �tubulins.17) Since P. capsici causes Phytophthora blight,a devastating disease of the pepper plant (C. annuum), itis worthwhile to note that � tubulin from P. capsici has86% sequence identity with CAnm �-TUB, and thatmutant sites for benzimidazole resistance are conservedin both. The two positions (Met200 and Thr323) that arenot significant for dinitroaniline resistance in E. indica �tubulins17) are conserved in C. annuum and P. capsici �tubulins also (Fig. 3 gray). These analyses are usefulduring the screening of non-phytotoxic, antimitoticagents for P. capsici.

Expression and purification of �=� tubulinProtein production from inclusion bodies is generally

protected from proteolytic degradation, and easilysolubilized, and contains almost pure protein in differentstates of aggregation, in an inactive form.31) In thisstudy, different concentrations of IPTG (0.1 to 1.0mM)were compared for their effectiveness in inducingmaximal expression of �=� tubulin. The optimal con-centration was 0.2mM (data not shown). After 12 h ofinduction with 0.2mM IPTG at 18 �C, recombinantplasmids pET28a �- and �-TUB exhibited high levels ofthe � and � tubulin overexpression respectively inE. coli BL21 (DE3), which accounted for about 50% oftotal cellular proteins (Fig. 4, lane AI). SDS–PAGEanalysis of the soluble and insoluble fractions of theinduced E. coli cell extracts indicated that the fusionprotein was mostly expressed as insoluble inclusionbodies (data not shown).During purification, there was no loss of protein

carrying the His6-tag in the flow-through, suggestingprotein binding to the nickel resin (data not shown).SDS–PAGE analysis of the fractions showed that elutionwith � 250mM imidazole resulted in a single band ofabout 55 kDa for � (Fig. 4A, lanes 6–12) and for �tubulin (Fig. 4B, lanes 6–10). This process typicallyyielded 2.0mg of � and 1.3mg of pure � tubulin from 1liter of culture. The procedure proved to be efficient, asreported from other sources such as tubulin from ginkgopollen, 2mg from 100 gm of ginkgo pollen,7) andMimosa pudica, 1.5mg from 50 g leaves.6) Even thoughdecreasing the temperature has been successful inreducing the tendency of certain proteins to aggre-gate,32,33) in this case, the content of soluble proteinremained unchanged at different induction times (6–15 h), growth phases, and through a temperature rangeof 30 to 18 �C (data not shown).

Confirmation of hetero-oligomer formationThe purified tubulin monomers were made to dimer-

ise, as described in ‘‘Materials and Methods’’ (Fig. 1).When analyzed by western blotting, the bands corre-sponding to dimeric tubulin reacted with both the anti-his and anti-T7 antibodies (Fig. 5A, B; lanes 3, 5).During the reaction of � and � tubulin in the presenceand absence of GTP, the fractions collected afterwashing of the resin did not show any band for �=�tubulin, indicating affinity binding of tubulin dimer toresin (Fig. 5A, B; lanes 2, 4). Since the elution of �tubulin only (T7þHis�) containing the reaction mixturedid not show any band (Fig. 5A, B; lane 1), it can beconcluded that there was no specific interaction between� tubulin and Niþ2 sepharose, and that � tubulin wasdimerized with � tubulin only.

Polymerization of tubulinContinuous changes in A350 were recorded during

polymerization of tubulin in the presence of GTP. Anobvious increase in absorbance was observed immedi-ately after the addition of GTP, reaching nearlysaturation point in 40min (Fig. 6). An increase in

1052 M.-H. JANG et al.

absorbance was recorded for � tubulin homodimers, butnot for � homodimers (data now shown). Such ��- and��-homodimers have been reported in recombinantnematode tubulin.34) Polymerization occurred at bothGTP concentrations tested and in the absence of GTP

(Fig. 6). There was a significant increase in polymer-ization at 2mM GTP concentration. In the absence ofGTP, tubulin produced abnormal structures due toaggregation of protein, without resulting in any domi-nant structure (data not shown). Haemonchus contortus

Fig. 3. The Amino Acid Sequence Alignment of C. annuum � Tubulin and Corresponding Sequences from Other Plants and Fungi.

Symbol meanings are as in Fig. 2. The amino acids highlighted in black/gray are discussed in the text. CAnm, C. annuum � tubulin

(ABP65322); ATha, A. thaliana (AAA32893); EInd, E. indica (AAD20178); PCap, P. capsicum (ABP65321); ANid, A. nidulans (1312295B);

NCra, N. crassa (AAA33617).

Cloning, Purification, and Polymerization of C. annuum Tubulin 1053

� and � tubulins produced abnormal structures in theabsence of GTP.34) Optimal formation of typical longmicrotubule-like structures occurred at � 1mg/ml ofeach tubulin concentration (Fig. 7A, B). This structureresembled previously published normal microtubularstructures from other plant sources in terms of appear-ance and size.6,7)

The correct and efficient refolding of an inclusionbody to its active form has been a topic in both basicresearch and industrial applications. This method gave�=� tubulin in correctly folded form since recombinanttubulins formed heterodimers and polymerized tomicrotubule like structures. In the future, this tubulin

from C. annuum can be used as control group forhigh-throughput screening of some fungicides againstP. capsici.

Acknowledgment

This study was carried out with the support of theOn-Site Cooperative Agriculture Research Project(20070201080024) and the National Institute of Agri-cultural Biotechnology, RDA, of the Republic of Korea.

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0.6

0.7

0 10 20 30 40 50 60 70 80 90

Time (min)

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(350

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)

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1054 M.-H. JANG et al.

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Cloning, Purification, and Polymerization of C. annuum Tubulin 1055