research article construction, expression, and...

Hindawi Publishing CorporationBioMed Research InternationalVolume 2013 Article ID 720285 6 pageshttpdxdoiorg1011552013720285

Research ArticleConstruction Expression and Characterization of ThymosinAlpha 1 Tandem Repeats in Escherichia coli

Xiao-Chang Xue Zhen Yan Wei-Na Li Meng Li Xin Qin Cun Zhang Qiang HaoZeng-Lu Wang Ning Zhao Wei Zhang and Ying-Qi Zhang

State Key Laboratory of Cancer Biology Department of Biopharmaceutics School of PharmacyFourth Military Medical University Xirsquoan 710032 China

Correspondence should be addressed to Ying-Qi Zhang xue xiaochangyahoocom

Received 11 June 2012 Revised 26 November 2012 Accepted 26 November 2012

Academic Editor Fabio Ferreira Perazzo

Copyright copy 2013 Xiao-Chang Xue et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

Thymosin alpha 1 (T1205721) which is composed of 28 amino acids has been commercializedworldwide for its immune-modulatory andantitumor effects T1205721 can stimulate T cell proliferation and differentiation from bone marrow stem cells augment cell-mediatedimmune responses and regulate homeostasis of immune system In this study we developed a novel strategy to produce T1205721concatemer (T1205721C) in Escherichia coli and compared its activity with chemically synthesized T1205721 Results showed that T1205721Ccan more effectively stimulate T cell proliferation and significantly upregulate IL-2 receptor expression We concluded that theexpression system for T1205721 concatemer was constructed successfully which could serve as an efficient tool for the production oflarge quantities of the active protein

1 Introduction

The tandem repeats of proteins and peptides are studiedwidely and formidable progress has been made in this fieldIt was reported that tandem amino acid repeats have manyfunctions of stabilizing proteins [1] maintaining conforma-tion [2] elevating activity and increasing half-life of proteinsin blood or tissues Frasch and colleagues suggested thattandem repeats present in Trypanosoma cruzi transsialidasestabilized the catalytic activity In addition repeats presenton T cruzi shed proteins increased trans-sialidase half-life inblood from 7 to almost 35 h [3] Some proteins that containtandemly repeated sequences play important roles in cellmembrane skeleton system [4 5]

Thymosin alpha1 (T1205721) is a heat-stable acidic polypeptidecomposed of 28 amino acid residues blocked at the N-terminus by an acetyl group [6 7] It is an immune modifierthat has been shown to trigger lymphocytes maturationaugment T cell function induce T-cell differentiation andpromote reconstitution of immune defects All these findingsshowed that T1205721 could be a useful restorative therapeuticagent in the treatment of immunodeficiency diseases andimmunosuppressed conditions [8ndash10]

In this study T1205721C which was composed of threerepeated copies of T1205721 was fuse-expressed with thioredoxin(trx) in E coli TOP10 strain and purified by heat treatmentand Q-Sepharose Fast Flow ion-exchange chromatographyThen T1205721C was released by treatment with 05M Cyanogenbromide (CNBr) and purified by SP-Sepharose Fast Flowchromatography In our strategy trx acts as a chaperonto help T1205721C folding and CNBr treatment removed anyexogenous amino acid (such as Met at the N-terminus fortranslation start) from T1205721C molecule So we can get theldquonaturalrdquo T1205721C Finally the biological activity of T1205721C on Tlymphocyte proliferation and IL-2R expression was assessed

2 Materials and Methods

21 Materials Restriction enzymes Taq DNA polymeraseand T4DNA ligase were purchased fromTaKaRa Expressionvector pThioHisA and E coli strain TOP10 (F-mcrAΔ(mrr-hsd RMS-mcrBC) 12059380 lacZΔM15 ΔlacX74 recA1 araΔ139Δ(ara-leu)7697 galU galK rpsL (Strr)endA1 nupG) were fromInvitrogen DNA fragments were synthesized in BIOASIASynthetic T1205721 (ZADAXIN) was from Sciclone Pharmaceuti-cals USA The anti-T1205721 antibody (ab55635) was purchased

2 BioMed Research International

pThioHisA Ligation

EcoRI

EcoRI

PstI

PstI

Ligation

1 2 1 2

3 4 3 4

P1 P1 P1

P2P2 P2

Annealing

PCR

Digestion

Am

p

LacI

q

ColE1

ColE1

ATG HP-thioredoxin EK site Tα1 3

3pThioHisA-Tα1

middot middot middot


Figure 1 Schematic diagram shows the strategy for constructing T1205721 concatemersThe Arabic numerals 1 to 4 are sequences 1 to 4 split fromT1205721 described previously for concatemers assembly P1 and P2 are forward and reverse primers for PCR

from Abcam and FITC-anti-IL-2R120573 (18344D 554452) wasfrom BD Pharmingen

22 T1205721C Gene Amplification T1205721C gene was cloned bygene synthesis and PCR (Figure 1) The forward primer(with an introduced EcoR I site) was p1 51015840-GGAATTCATG-TCTGATGCAGCCGTGGACACCAGCAGCG-31015840 and thereverse primer (with an introduced Pst I site) was p2 51015840-GCACTGCAGTCAGTTCTGGGCCTCCTCCACCACCT-31015840 The template for PCR was annealing products of 4synthesized fragments listed in Table 1 PCR product wascloned into pGEM-3Zf plasmid to obtain vector pGEM-T1205721C for identification

23 Construction of Expression Vector pThioHisA-T1205721C Thevector pGEM-T1205721C was digested with EcoR I and Pst I andcloned into expression vector pThioHisA digested with thesame enzymesThe candidate plasmid pThioHisA-T1205721Cwasthen confirmed by restriction enzymes digestion and DNAsequencing

24 Expression of the Fusion Protein The plasmid pThioHi-sA-T1205721C was transformed into E coli TOP10 strain A singlecolonywas inoculated into 10mLLuria-Bertani (LB)mediumsupplemented with ampicillin (100 120583gmL) and grown at200 rpm and 37∘C overnight Then it was inoculated into 300mL fresh LB medium in a 500 mL shake flask and cultureduntil the OD600 reached 05 Trx-T1205721C expression wasinduced by 1mM IPTG (final concentration) for 4 h Large

Table 1 Nucleotide sequence of DNA fragments split from T1205721 forconcatemers assembly

Number nucleotide sequence

1 GACACCAGCAGCGAGATCACCACCAAGGACCGGAAGGAGAAGAAGGAGGTGGTG

2 GAGGAGGCCGAGAACAGCGACGCCGCCGTG

3 CTTGGTGGTGATCTCGCTGCTGGTGTCCACGGCGGCGTCGCT

4 GTTCTCGGCCTCCTCCACCACCTCCTTCTTCTCCTTCAGGTC

scale fed-batch culture was performed in a 5-L fermentor aspreviously described [11]

25 Purification of T1205721C Cell pellet was suspended in20mM TrisHCl buffer (pH 80) in proportion of 200 gLand disrupted by sonication Then the lysate was incubatedat 80∘C for 10min (shaken once every 2-3min) and cooledquickly Samples were centrifuged at 12 000 g for 20minand the supernatant was loaded onto a Q-Sepharose FastFlow chromatography column and eluted with linear NaClgradient The purified Trx-T1205721C was then cleaved by CNBr(05M) in 70 formic acid for 24 h The cleavage reactionwas stopped by addition of ten times amount of H

2O [12] and

T1205721C was purified by SP-Sepharose Fast Flow chromatogra-phy Purified T1205721C was dialyzed against PBS for later use

BioMed Research International 3

(kD

a)

321

974

662

43

31

201

1 2

1 5432

16949

14404

107

8159

(kD

a)

321

(a)

(b) (c)

(d)

(e)(f)

Figure 2 Expression purification and identification of T1205721C (a) Expression of trx-T1205721C in TOP10 Lane 1 protein marker lane 2 totalbacterial proteins of pThioHisA-T1205721CTOP10 without induction lane 3 total bacterial protein with IPTG induction (b) Chromatogram ofQ-Sepharose Fast Flow chromatography for purification of trx-T1205721C The arrow indicated trx-T1205721C (c) Chromatogram of SP-SepharoseFast Flow chromatography for purification of T1205721C The arrow indicated T1205721C (d) SDS-PAGE analysis of trx-T1205721C purification Lane 1ndash3total proteins of pThioHisA-T1205721CTOP10 after IPTG induction (1 h 3 h 5 h) lane 4 supernatant of lysate heated at 80∘C for 10min lane 5purified trx-T1205721C (e) Tricine-SDS-PAGE analysis of T1205721C purification Lane 1 standard peptide marker lane 2 cleavage products withoutT1205721C lane 3 purified T1205721C (f) Western-blot analysis of trx-T1205721C Lane 1 total bacterial proteins after IPTG induction lane 2 purifiedtrx-T1205721C

26 Western-Blot Analysis Proteins were transferred tonitrocellulose membranes (022 120583m Invitrogen) after SDS-PAGE using a Bio-Rad Semi-Dry electrophoretic cell West-ern blot analyses were carried out using a T1205721 specificantibody and followed by a phosphatase-conjugated goatanti-mouse IgG (Boster China) Western Blue StabilizedSubstrate (Promega) for alkaline phosphatase was used forvisualization

27 Biological Activity Assay The proliferation response ofsplenocytes was determined by MTT assay Spleens fromC57BL6mice were dispersed through nylonmesh to generatea single-cell suspensionThen lymphocytes were separated byEZ-Sep 1times Lymphocyte SeparationMedium (DKW33-R0100Dakewe Biotech Company China) and suspended at 4 times106mL in RPMI 1640 media For proliferation assay cellswere seeded in 96-well plates (4 times 105well) and culturedin the presence of 25120583gmL concanavalin A (ConA) at37∘C in 5 CO

2in humid air Six h later 90120583L of T1205721C

diluted with RPMI 1640 media was added to all but thecontrol wells The synthetic T1205721 and media were used aspositive and negative controls After 66 h incubation 20120583Lof MTT (05mgmL) solution was added and the plates were

centrifuged (2000 rpm 25∘C 10min) 4 h later Supernatantswere discarded and 100120583L of DMSO was added Afterincubated at room temperature for 10min the solubilizedreduced MTT was measured at 570 nm using a Bio-Radplate reader and the optical densities were used for calculategrowth rate with the formula

Growth rate () =OD sampleOD control

times 100 (1)

To evaluate the effect of T1205721C on the expression level ofIL-2R on T lymphocytes cells were isolated as before andcultured in the presence of ConA and T1205721C The syntheticT1205721 and a recombinant T1205721 monomer prepared in our labwere used as positive controls Cells were collected andstained 48 h later according to standard protocol In brief 5 times105 cells were washed with PBS and stained in ldquoFACS bufferrdquo(PBS with 01 sodium azide 2 FBS and 1120583M EDTA)with FITC-anti-mIL-2R120573 for 10min at room temperatureAfter washing cells were fixed for 30 minutes on ice with4 paraformaldehyde and analyzed on a FACSCalibur flowcytometer (BD Biosciences)


60

80

100

120

140

160

180

200

5 10 20 40

Gro

wth

rat

e (

)

Concentration (μgmL)

Control

lowast

lowastlowastlowastlowast

lowastlowast

lowastlowast

Tα1

Tα1 3

Figure 3 T1205721C stimulated proliferation ofmouse spleen lymphocytes T lymphocytes fromB6mice spleen were treated with ConA (control)or ConA plus T1205721C or synthetic T1205721 Cell proliferation was determined by the MTT viability assay The assays were repeated in triplicate(lowast119875 lt 005 compared with control grouplowastlowast119875 lt 005 compared with T1205721 group)

3 Results and Discussion

31 T1205721C Gene Cloning Although synthetic T1205721 has beensuccessfully applied in clinical trials for immunodeficiencydiseases therapy the high costs is still a hard nut to crackFortunately molecular biology techniques allowed us toproduce recombinant T1205721 in E coli Considering that T1205721 istoo small to be directly expressed in E coli it was usuallyassembled as concatemers But some exogenous amino acidresidues such as His6 tag or methionine (Met) introducedby the initiation codon AUG usually affects the effect ofconcatemers [13]

In order to produce the real ldquonaturalrdquo concatemers of T1205721we put forward a new strategy as showed in Figure 1 By thisstrategy we obtained a series of T1205721 concatemers in whichT1205721C that was assembled by three repeated copies of T1205721gene owned highest proportion After cloning into pGEM-3Zf vector the gene was proven by enzyme digestion andDNA sequencingThe sequence of T1205721C gene was consistentwith our design as follows 51015840-atgagcgacgccgccgtggacaccagc-agcgagatcaccaccaaggaccggaaggagaagaaggaggtggtggaggag-gccgagaacagcgacgccgccgtggacaccagcagcgagatcaccaccaaggac-cggaaggagaagaaggaggtggtggaggaggccgagaacagcgacgccgcc-gtggacaccagcagcgagatcaccaccaaggaccggaaggagaagaaggaggtg-gtggaggaggccgagaactga-31015840

32 Expression of Recombinant Fusion Protein Both SDS-PAGE (Figure 2(a)) and Western blot (Figure 2(f)) analysesof the induced supernatant from pThioHisA-T1205721CTOP10showed that a new 31 kDa protein which can be specificallyrecognized by T1205721 antibody was produced It suggested thattrx-T1205721C was successfully expressed Trx was used as a

chaperon to guarantee the correct folding of T1205721C and trx-T1205721C was expressed as a soluble fusion protein

33 Purification of T1205721C Both trx and T1205721 are heat-stableproteins so trx-T1205721C was easily purified by one-step Q-Sepharose Fast Flow chromatography after the lysate ofrecombinant bacterial cells was heated at 80∘C for 10min(Figures 2(b) and 2(d)) Then the purified trx-T1205721C wascleaved by CNBr and T1205721C was purified by SP-SepharoseFast Flow chromatography (Figures 2(c) and 2(e)) 2L-Tricine-SDS-PAGE [14] and HPLC analyses were used toidentify the purity of T1205721C CNBr treatment was utilizedhere to remove the redundant Met from the N-terminus ofT1205721C to obtain the real ldquonaturalrdquo molecule

34 Biological Activity of T1205721C We expected that the tan-dem repeats could obtain stronger activity through elongat-ing the half-life of T1205721 and simulating polymerization ofmonomer molecules and thereafter triggering the polymer-ization and activation of receptors which was usually used bymolecules to gain function

To examine the effect of T1205721C on stimulating the pro-liferation of splenic lymphocytes we compared the prolif-eration ratio of mice lymphocytes treated with syntheticT1205721 ZADAXIN and T1205721C MTT assay results showed that40 120583gmL synthetic T1205721 could induce significant proliferationof lymphocytes compared to the control (119875 lt 005) whereas5 120583gmL T1205721C could induce significant proliferation (119875 lt005) Furthermore the effect of 10120583gmLT1205721Cwas strongerthan that of 40 120583gmL synthetic T1205721 (Figure 3)

In addition the upregulation of IL-2R on lymphocytes byZADAXIN purified recombinant T1205721 monomer and T1205721Cwas compared Results showed that when costimulated with


1 10 100 1000

575 nmG ssvsFSC

oun

t

01

FT1

(a)

01 1 10 100 1000

575 nmG ssvsFS

Cou

nt

FT1

(b)

1 10 100 1000

575 nmG ssvsFS

Cou

nt

01

FT1

(c)

01 1 10 100 1000

575 nmG ssvsFS

Cou

nt

FT1

(d)

G ssvsFS

Cou

nt

1 10 100 1000

575 nm

01

FT1

(e)

Figure 4 Effect on upregulation of IL-2R expression level on T cell surface of T1205721C T lymphocytes from B6 mice spleen were cultured inthe presence of ConA (b) or ConA plus standard synthesized T1205721 (c) recombinant T1205721 (d) and recombinant T1205721C (e) respectively (a) Theunstrained control Data are representative of three experiments

ConA IL-2R expression level on T cell was upregulated byall these three molecules and T1205721C obtained strongest effect(Figure 4)

4 Conclusions

Trx-T1205721C was expressed in E coli as a soluble form andthe real ldquonaturalrdquo T1205721C was conveniently purified by heattreatment and ion-exchange chromatography As expectedthe bioactivity of T1205721C was stronger than that of syntheticT1205721 Lower dose (5120583gmL) of T1205721C apparently stimulated

the proliferation of T lymphocytes compared with thatof ZADAXIN (40 120583gmL) In addition T1205721C significantlyupregulated IL-2R onT cell which is very important for T cellactivation and proliferation in vivo The detailed mechanismfor stronger effect of T1205721C and the pharmacokinetics ofdifferent tandem repeats are still under investigation

Acknowledgment

This project was supported by the Natural Science Fund ofChina (Project no 31000406)


References

[1] R I MacDonald and E V Pozharski ldquoFree energies of urea andof thermal unfolding show that two tandem repeats of spectrinare thermodynamically more stable than a single repeatrdquoBiochemistry vol 40 no 13 pp 3974ndash3984 2001

[2] J Varea J L Saiz C Lopez-Zumel et al ldquoDo sequence repeatsplay an equivalent role in the choline-binding module of pneu-mococcal LytA amidaserdquo Journal of Biological Chemistry vol275 no 35 pp 26842ndash26855 2000

[3] C A Buscaglia J Alfonso O Campetella and A C C FraschldquoTandem amino acid repeats from Trypanosoma cruzi shedantigens increase the half-life of proteins in bloodrdquo Blood vol93 no 6 pp 2025ndash2032 1999

[4] A Schneider A Hemphill T Wyler and T Seebeck ldquoLargemicrotubule-associated protein of T brucei has tandemlyrepeated near-identical sequencesrdquo Science vol 241 no 4864pp 459ndash462 1988

[5] P C Cotrim G Paranhos-Baccala M R Santos et al ldquoOrgan-ization and expression of the gene encoding an immunodom-inant repetitive antigen associated to the cytoskeleton of Try-panosoma cruzirdquo Molecular and Biochemical Parasitology vol71 no 1 pp 89ndash98 1995

[6] A L Goldstein T L LowMMcAdoo et al ldquoThymosin alpha 1isolation and sequence analysis of an immunologically activethymic polypeptiderdquo Proceedings of the National Academy ofSciences of the United States of America vol 74 pp 725ndash7291977

[7] T L K Low and A L Goldstein ldquoThe chemistry and biology ofthymosin II Amino acid sequence analysis of thymosin 1205721 andpolypeptide 1205731rdquo Journal of Biological Chemistry vol 254 no 3pp 987ndash995 1979

[8] F Salvati G Rasi L Portalone A Antilli and E Garaci ldquoCom-bined treatment with thymosin-alpha1 and low-dose interfer-on-alpha after ifosfamide in non-small cell lung cancer a phase-II controlled trialrdquo Anticancer Research vol 16 no 2 pp 1001ndash1004 1996

[9] SMoscarella G Buzzelli R G Romanelli et al ldquoInterferon andthymosin combination therapy in naive patients with chronichepatitis C preliminary resultsrdquo Liver vol 18 no 5 pp 366ndash369 1998

[10] F Pica M Fraschetti C Matteucci C Tuthill and G RasildquoHigh doses of thymosin alpha 1 enhance the anti-tumor effi-cacy of combination chemo-immunotherapy for murine B16melanomardquo Anticancer Research vol 18 no 5 pp 3571ndash35781998

[11] X Xue Z Wang Z Yan J Shi W Han and Y Zhang ldquoProduc-tion and purification of recombinant human BLyS mutant frominclusion bodiesrdquo Protein Expression and Purification vol 42no 1 pp 194ndash199 2005

[12] J Kim J M Park and B J Lee ldquoHIGH-level expression andefficient purification of the antimicrobial peptide gaegurin 4 inE colirdquo Protein and Peptide Letters vol 4 no 6 pp 391ndash3961997

[13] Y Chen L Zhao G Shen et al ldquoExpression and analysis ofthymosin 1205721 concatemer in Escherichia colirdquo Biotechnology andApplied Biochemistry vol 49 no 1 pp 51ndash56 2008

[14] J H Shi Y T Zhao J LWangWHan Z Yan and Y Q ZhangldquoAnalysis of low molecular peptides by sodium dodecyl sulfate-polyacrylamide gel electrophoresisrdquo Journal of the Fourth Mili-tary Medical University vol 21 no 6 pp 761ndash763 2000

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Volume 2014

Stem CellsInternational



Enzyme Research



Microbiology


pThioHisA Ligation

EcoRI

EcoRI

PstI

PstI

Ligation

1 2 1 2

3 4 3 4

P1 P1 P1

P2P2 P2

Annealing

PCR

Digestion

Am

p

LacI

q

ColE1

ColE1

ATG HP-thioredoxin EK site Tα1 3

3pThioHisA-Tα1



Figure 1 Schematic diagram shows the strategy for constructing T1205721 concatemersThe Arabic numerals 1 to 4 are sequences 1 to 4 split fromT1205721 described previously for concatemers assembly P1 and P2 are forward and reverse primers for PCR

from Abcam and FITC-anti-IL-2R120573 (18344D 554452) wasfrom BD Pharmingen

22 T1205721C Gene Amplification T1205721C gene was cloned bygene synthesis and PCR (Figure 1) The forward primer(with an introduced EcoR I site) was p1 51015840-GGAATTCATG-TCTGATGCAGCCGTGGACACCAGCAGCG-31015840 and thereverse primer (with an introduced Pst I site) was p2 51015840-GCACTGCAGTCAGTTCTGGGCCTCCTCCACCACCT-31015840 The template for PCR was annealing products of 4synthesized fragments listed in Table 1 PCR product wascloned into pGEM-3Zf plasmid to obtain vector pGEM-T1205721C for identification

23 Construction of Expression Vector pThioHisA-T1205721C Thevector pGEM-T1205721C was digested with EcoR I and Pst I andcloned into expression vector pThioHisA digested with thesame enzymesThe candidate plasmid pThioHisA-T1205721Cwasthen confirmed by restriction enzymes digestion and DNAsequencing

24 Expression of the Fusion Protein The plasmid pThioHi-sA-T1205721C was transformed into E coli TOP10 strain A singlecolonywas inoculated into 10mLLuria-Bertani (LB)mediumsupplemented with ampicillin (100 120583gmL) and grown at200 rpm and 37∘C overnight Then it was inoculated into 300mL fresh LB medium in a 500 mL shake flask and cultureduntil the OD600 reached 05 Trx-T1205721C expression wasinduced by 1mM IPTG (final concentration) for 4 h Large

Table 1 Nucleotide sequence of DNA fragments split from T1205721 forconcatemers assembly

Number nucleotide sequence

1 GACACCAGCAGCGAGATCACCACCAAGGACCGGAAGGAGAAGAAGGAGGTGGTG

2 GAGGAGGCCGAGAACAGCGACGCCGCCGTG

3 CTTGGTGGTGATCTCGCTGCTGGTGTCCACGGCGGCGTCGCT

4 GTTCTCGGCCTCCTCCACCACCTCCTTCTTCTCCTTCAGGTC

scale fed-batch culture was performed in a 5-L fermentor aspreviously described [11]

25 Purification of T1205721C Cell pellet was suspended in20mM TrisHCl buffer (pH 80) in proportion of 200 gLand disrupted by sonication Then the lysate was incubatedat 80∘C for 10min (shaken once every 2-3min) and cooledquickly Samples were centrifuged at 12 000 g for 20minand the supernatant was loaded onto a Q-Sepharose FastFlow chromatography column and eluted with linear NaClgradient The purified Trx-T1205721C was then cleaved by CNBr(05M) in 70 formic acid for 24 h The cleavage reactionwas stopped by addition of ten times amount of H

2O [12] and

T1205721C was purified by SP-Sepharose Fast Flow chromatogra-phy Purified T1205721C was dialyzed against PBS for later use


(kD

a)

321

974

662

43

31

201

1 2

1 5432

16949

14404

107

8159

(kD

a)

321

(a)

(b) (c)

(d)

(e)(f)








times 100 (1)



60

80

100

120

140

160

180

200

5 10 20 40

Gro

wth

rat

e (

)


Control

lowast


lowastlowast

lowastlowast

Tα1

Tα1 3












1 10 100 1000

575 nmG ssvsFSC

oun

t

01

FT1

(a)

01 1 10 100 1000

575 nmG ssvsFS

Cou

nt

FT1

(b)

1 10 100 1000

575 nmG ssvsFS

Cou

nt

01

FT1

(c)

01 1 10 100 1000

575 nmG ssvsFS

Cou

nt

FT1

(d)

G ssvsFS

Cou

nt

1 10 100 1000

575 nm

01

FT1

(e)



4 Conclusions



Acknowledgment



References






















Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


(kD

a)

321

974

662

43

31

201

1 2

1 5432

16949

14404

107

8159

(kD

a)

321

(a)

(b) (c)

(d)

(e)(f)








times 100 (1)



60

80

100

120

140

160

180

200

5 10 20 40

Gro

wth

rat

e (

)


Control

lowast


lowastlowast

lowastlowast

Tα1

Tα1 3












1 10 100 1000

575 nmG ssvsFSC

oun

t

01

FT1

(a)

01 1 10 100 1000

575 nmG ssvsFS

Cou

nt

FT1

(b)

1 10 100 1000

575 nmG ssvsFS

Cou

nt

01

FT1

(c)

01 1 10 100 1000

575 nmG ssvsFS

Cou

nt

FT1

(d)

G ssvsFS

Cou

nt

1 10 100 1000

575 nm

01

FT1

(e)



4 Conclusions



Acknowledgment



References






















Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


60

80

100

120

140

160

180

200

5 10 20 40

Gro

wth

rat

e (

)


Control

lowast


lowastlowast

lowastlowast

Tα1

Tα1 3












1 10 100 1000

575 nmG ssvsFSC

oun

t

01

FT1

(a)

01 1 10 100 1000

575 nmG ssvsFS

Cou

nt

FT1

(b)

1 10 100 1000

575 nmG ssvsFS

Cou

nt

01

FT1

(c)

01 1 10 100 1000

575 nmG ssvsFS

Cou

nt

FT1

(d)

G ssvsFS

Cou

nt

1 10 100 1000

575 nm

01

FT1

(e)



4 Conclusions



Acknowledgment



References






















Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


1 10 100 1000

575 nmG ssvsFSC

oun

t

01

FT1

(a)

01 1 10 100 1000

575 nmG ssvsFS

Cou

nt

FT1

(b)

1 10 100 1000

575 nmG ssvsFS

Cou

nt

01

FT1

(c)

01 1 10 100 1000

575 nmG ssvsFS

Cou

nt

FT1

(d)

G ssvsFS

Cou

nt

1 10 100 1000

575 nm

01

FT1

(e)



4 Conclusions



Acknowledgment



References






















Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


References






















Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology








Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

research article construction, expression, and...

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