research article enhancement of polymerase activity of the...

Research ArticleEnhancement of Polymerase Activity of the Large Fragment inDNA Polymerase I from Geobacillus stearothermophilus bySite-Directed Mutagenesis at the Active Site

Yi Ma1 Beilei Zhang1 MengWang1 Yanghui Ou1 JufangWang1 and Shan Li12

1School of Bioscience amp Bioengineering South China University of Technology Guangzhou 510006 China2Guangdong Province Key Laboratory of Fermentation and Enzyme Engineering South China University of TechnologyGuangzhou 510006 China

Correspondence should be addressed to Shan Li lishanscuteducn

Received 25 August 2016 Revised 13 October 2016 Accepted 19 October 2016

Academic Editor Sujit S Jagtap

Copyright copy 2016 Yi Ma et al This is an open access article distributed under the Creative Commons Attribution License whichpermits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

The large fragment of DNA polymerase I from Geobacillus stearothermophilus GIM1543 (Bst DNA polymerase) with 51015840-31015840 DNApolymerase activity while in absence of 51015840-31015840 exonuclease activity possesses high thermal stability and polymerase activity BstDNA polymerase was employed in isothermal multiple self-matching initiated amplification (IMSA) which amplified the interestsequence with high selectivity and was widely applied in the rapid detection of human epidemic diseases However the detailedinformation of commercial Bst DNA polymerase is unpublished and well protected by patents which makes the high price ofcommercial kits In this study wild-type Bst DNA polymerase (WT) and substitution mutations for improving the efficiency ofDNA polymerization were expressed and purified in E coli Site-directed substitutions of four conserved residues (Gly310 Arg412Lys416 and Asp540) in the activity site of Bst DNA polymerase influenced efficiency of polymerizing dNTPs The substitution ofresidue Gly310 by alanine or leucine and residue Asp540 by glutamic acid increased the efficiency of polymerase activity All mutantswith higher polymerizing efficiency were employed to complete the rapid detection of EV71-associated hand foot and mouthdisease (HFMD) by IMSA approach with relatively shorter period which is suitable for the primary diagnostics setting in rural andunderdeveloped areas

1 Introduction

Nucleic acid amplification techniques are widely used indiagnosis of infectious diseases genetic traits and otherclinical medics in application-oriented fields However thetraditional PCR procedure is time consuming and requiresexpensive special equipment which is rarely available inrural and underdeveloped areas Recently Notomi et aldeveloped a rapid detection of nucleic acid bases loop-mediated isothermal amplification (LAMP) which withhigh sensitivity and specificity successfully accomplished theamplification of the target DNA sequence at constant tem-perature in water bath for one hour neglecting normal three-temperature cycles [1 2] LAMP technique has been widelyused in the virus detection for infectious diseases such asavian influenza [3] HFMD [4] Dengue Virus [5] the human

immunodeficiency virus [6] and Ebola virus [7] Presentlya novel and simple colorimetric isothermal multiple self-matching initiated amplification (IMSA) was developed toachieve rapid detection of EV71 in the early phase of HFMDthrough visual color change by addition of hydroxyl naphtholblue dye [8] Similar to the LAMP assay the IMSA assay isan in vivo nucleic acid amplification technique depending onBst DNA polymerase dNTPs magnesium ion betaine andthree pairs of primers consisting of one pair of stem primers(SteF and SteR) one pair of outer primers (DsF and DsR)and one pair of inner primers (FIT and RIT) Comparedto the LAMP assay primer design for IMSA assay has itsown distinct features which significantly reduce the detectionlimits of LAMP assay In order to make quantitative analysisaccurately the IMSA assay generated a fluorescence increasein positive samples allowing real-time monitoring detection

Hindawi Publishing CorporationBioMed Research InternationalVolume 2016 Article ID 2906484 8 pageshttpdxdoiorg10115520162906484

2 BioMed Research International

Since Kornberg discovered and studied DNA polymeraseI in detail from Escherichia coli in 1958 [9] various DNApolymerases such as DNA polymerases 120572 and 120573 and RNApolymerase have been isolated and characterized from bothprokaryotic and eukaryotic organisms [10 11] The indis-pensable Bst DNA polymerase I in the isothermal ampli-fication was widely applied and played a key role in clin-ical rapid detection of virus infections Similar to otherDNA polymerase I Bst DNA polymerase from Bacillusstearothermophilus consisted of three independent domainswhich perform biological function individually (I) 51015840-31015840exonuclease activity (II) 51015840-31015840 DNA polymerase activity and(III) 31015840-51015840 exonuclease activity [12] The domains II andIII are located in the C-terminus of Bst DNA polymerasedesignated as the large fragment (LF) [13] The full-lengthof Bst DNA polymerase I gene consisted of 2634-base-pairwhile the truncated LF gene devoid of the 51015840-terminal 876-base-pair could be translated into a predicated 671 kD DNApolymerase with C-terminal poly (His)6 which lacks theinherent 51015840-31015840 proofreading exonuclease activity and wasextremely efficient in polymerizing dNTPs in PCR [14]Since Bst DNA polymerase meets all requirements for highsensitivity polymerization activity and thermal stability itis extensively applied in isothermal amplification procedureof rapid detection However Bst DNA polymerase is onlyavailable popular in some international companies as NewEnglandBiolabs at high priceTherefore it is urgent to designproduce and purify a new and efficient Bst DNA polymeraseto satisfy growing demand of less developed area In thisstudy we cloned the gene of Bst DNA polymerase I froma new strain (Geobacillus stearothermophilus GIM1543) andmake various site-directed substitutions of four conservedresidues in the polymerase active site which is important inbinding the DNA primer terminus and dNTP to catalyze thepolymerase reaction [15 16] Three mutants showed higherpolymerization activity than the commercial Bst DNA poly-merase and consequently would lay the roots for promotingits wide application

2 Materials and Methods

21 Strains Plasmids Enzymes and Reagents Geobacillusstearothermophilus was purchased from Microbial CultureCollection Center of Guangdong Institute of MicrobiologyChina Competent E coli cells DH5120572 and BL21 (DE3) werepurchased fromTIANGENBiotech (Beijing China) PlasmidpET21a was preserved by our own laboratory Restrictionendonucleases Nde I and Xho I were purchased from Ther-moFisher Scientific Co Ltd (Shanghai China) Reagentsrelated to Bst DNA pol LF gene cloning and site-directedmutagenesis were purchased from TaKaRa BiotechnologyCo Ltd (Dalian China) The HisTrapFF column from GEHealthcare (Piscataway NJ USA) was used for proteinspurification The chemicals used for HPLC were obtainedfrom Sigma-Aldrich Chemical (St Louis USA) All chemi-cals used in this study were of analytical grade

22 Construction of Expression Vector Genome DNA fromGeobacillus stearothermophilus was prepared with a genome

extraction kit (Sangon Shanghai China) according to man-ufacturerrsquos handbook The coding region of LF in BstDNA polymerase (Bst pol LF) using PrimeSTAR HS DNApolymerase was amplified with the following primers theforward primer was 51015840-CTGTTCCATATGGAAGGCGAA-AAGCCGCTCGCC-31015840 and the reverse primer was 51015840-CCGCTCGAGTTTGGCGTCGTACCACGTC-31015840 Restric-tion sites Nde I and Xho I used for subsequent amplificationare shown in bold and underline PCR was implementedunder the following condition initial heating at 95∘C for5min 30 cycles of 95∘C for 30 s 56∘C for 1min and 72∘Cfor 10min followed by an extension step at 72∘C for 5minThe PCR product of 1758 bp DNA fragment was separated by15 agarose gel electrophoresis and purified using a DNA gelextraction kit (Sangon Shanghai) Purified PCR product wasdigested with Nde I and Xho I and ligated into pET21a vector(Novagen Madison) yielding the plasmid pET21a-Bst-LF-H6with one poly (His)6 tag at C-terminus and transformed intoE coli DH5120572 cells Recombinant DH5120572 cells were spreadon LuriandashBertani (LB) agar plates containing 50 ng120583L ofkanamycin and incubated at 37∘C overnight The positiveclones were confirmed by colony PCR and DNA sequenceanalysis The sequence similarity of Bst DNA pol LF waschecked by the BLAST program

23 Mutagenesis Experiment Multiple protein sequencesincluding WT target protein sequence were aligned and ana-lyzed Several amino acid residues were selected as mutationsites shown in Table 1 Using theWT gene of Bst DNA pol LFas template the Restriction Free (RF) cloning PCR method[17] was applied to construct the mutated genes with primerpairs shown in Table 1 The Bst DNA pol LF proteins wereexpressed with C-terminal 6x His-tag promoting proteinspurification All purified proteins (WT and mutants) and thecommercial Bst 20 DNA polymerase were evaluated by SDS-PAGE

24 Expression and Purification of the WT and MutantDerivatives of Bst DNA pol LF Plasmid pET21a-Bst-LF-H6 was transformed into E coli BL21 (DE3) (NovagenMadison WI USA)The recombinant bacteria were inducedto express recombinant LF-Bst-H6 by adding isopropyl-120573-D-thiogalactopyranoside (IPTG) at a final concentration of1mM to a culture with OD600 of approximately 06ndash08 andincubating at 37∘C for 6 h Cell culture (1 L) was harvestedby centrifugation at 5000timesg for 30min The cell pelletswere resuspended in 40mL 1x PBS buffer (50mM Tris-HCland 300mM NaCl) and lysed with 450 sonication pulses(400W 3 s with a 5 s interval) cooled in ice water bath Thesuspension was centrifuged (11000timesg at 4∘C for 30min) andpassed through a 022 120583m filter followed by applying to a 5mL HiTrapFF chelating HP column The target protein Bst-LF-H6 was purified using the standard nickel affinity chro-matography procedure and washed with 5 column volumeseach of 50mM 100mM 150mM and 200mM imidazole incolumn buffer (20mM Tris-HCl 500mM NaCl and 20glycerol pH 80) followed by gel-filtration on Bio-Gel P-6(Bio-Rad Laboratories Inc) according to the manufacturerrsquos

BioMed Research International 3

Table 1 Oligonucleotides used in substitution mutagenesis for 10 residues of Bst DNA pol LF

Mutagenesis Primer name Sequence

G310A F (51015840-31015840) GTGGTGCACCCCGTGACGGCGAAAGTGCACACGATGTTCAATCAGR (51015840-31015840) CTGATTGAACATCGTGTGCACTTTCGCCGTCACGGGGTGCACCAC

G310L F (51015840-31015840) GTGGTGCACCCCGTGACGCTCAAAGTGCACACGATGTTCAATCAGR (51015840-31015840) CTGATTGAACATCGTGTGCACTTTGAGCGTCACGGGGTGCACCAC

D540A F (51015840-31015840) CGCCTGTTGCTGCAAGTGCATGCAGAACTGATTTTGGAGGCGCCGAAAGAR (51015840-31015840) CTCTTTCGGCGCCTCCAAAATCAGTTCTGCATGCACTTGCAGCAACAGGCG

D540E F (51015840-31015840) CGCCTGTTGCTGCAAGTGCATGAAGAACTGATTTTGGAGGCGCCGAAAGAR (51015840-31015840) CTCTTTCGGCGCCTCCAAAATCAGTTCTTCATGCACTTGCAGCAACAGGCG

R412A F (51015840-31015840) CGAAGAAGACGTGACAGCCAACATGCACGCCAAGCGAAGGCCGTCAATTR (51015840-31015840) AATTGACGGCCTTCGCTTGGCGTGCCATGTTGGCTGTCACGTCTTCTTCG

R412E F (51015840-31015840) CGAAGAAGACGTGACAGCCAACATGGAACGCCAAGCGAAGGCCGTCAATTR (51015840-31015840) AATTGACGGCCTTCGCTTGGCGTTCCATGTTGGCTGTCACGTCTTCTTCG

K416A F (51015840-31015840) GTGACAGCCAACATGCGCCGCCAAGCGGCAGCCGTCAATTTTGGCAR (51015840-31015840) TGCCAAAATTGACGGCTGCCGCTTGGCGGCGCATGTTGGCTGTCAC

K416D F (51015840-31015840) GTGACAGCCAACATGCGCCGCCAAGCGGATGCCGTCAATTTTGGCAR (51015840-31015840) TGCCAAAATTGACGGCATCCGCTTGGCGGCGCATGTTGGCTGTCAC

G310A-D540E F (51015840-31015840) GTGGTGCACCCCGTGACGGCGAAAGTGCACACGATGTTCAATCAGR (51015840-31015840) CTGATTGAACATCGTGTGCACTTTCGCCGTCACGGGGTGCACCAC

G310L-D540E F (51015840-31015840) GTGGTGCACCCCGTGACGCTCAAAGTGCACACGATGTTCAATCAGR (51015840-31015840) CTGATTGAACATCGTGTGCACTTTGAGCGTCACGGGGTGCACCAC

Underlined sequences indicate the mutant codons

Table 2 Oligonucleotides used in IMSA assay

Primer name SequenceDsF-EV71 5-ACCATTGATAAGCACTCGCAGGGTCAAGCTGTCAGACCCTCC-3DsR-EV71 5-GAACACAAACAGGAGAAAGATCTTGTGAGAACGTGCCCATCA-3FIT-EV71 5-TCCGAATGTGGGATATCCGTCATAAGTTTCAGTGCCATTCATGTC-3RIT-EV71 5-TTATGACGGATATCCCACATTCGGAAGGACATGCCCCGTATT-3SteF-EV71 5-GAACACAAACAGGAGAAAGATCTTG-3SteR-EV71 5-ACCATTGATAAGCACTCGCAGG-3

instructions Bst DNA pol LF samples were collected andstored in buffer (20mMNa-phosphate buffer pH 74 50mMNaCl) All fractions were dissolved in SDS sample buffer andloaded on 12 (wv) sodium dodecyl sulfate polyacrylamidegel electrophoresis (SDS-PAGE) and stained with CoomassieBlue The concentrations were determined by BCA ProteinAssay Kit (Sangon Shanghai China) Fractions of the elutewere stored in store buffer (20mM Tris-HCl 50mM NaCland 20 glycerin pH 80) at minus80∘C

25 Enzymatic Activity and Protein Assays The VP1 geneat genome positions (nucleotides 2978 to 3248) in EV71subgenotype C4 isolate (GenBank accession numberGQ2793701) from Chinese Center for Disease Control wasselected as reference virus The primers for the IMSA assaywas designedwith the aid of Primer Explorer V4 [8] shown inTable 2 and synthesized by Huada Gene Co Ltd (ShenzhenChina) The optimal condition of IMSA assay was carried

out in a 25 120583L volume containing the components 125 120583L of2x isothermal reaction mixture consisted of reaction bufferand dNTPs (Deaou Biotechnology Guangzhou China)10 120583L of each primer (DsF and DsR 50mM FIT and RIT200mM and SteF and SteR 400mM) 03 120583L of EV71 DNAtemplate and 10120583L of commercial Bst 20 DNA polymerase(8U120583L New England Biolabs MA USA) as positive controlwhile the other tubes contained equal amounts of WTor mutant proteins finally adding ddH2O up to 25120583L inreaction tubes The reactions were evaluated in two differentways The first assay was the visual IMSA assay by adding10 hydroxynaphthol blue (HNB) dye to the mixture beforeamplification and the other assay was performed in a Deaou-308C constant temperature fluorescence detector (DeaouBiotechnology Guangzhou China) with the additionof 10 120583L diluted SYTO9 fluorescent nucleic acid (LifeTechnologies Gaithersburg USA) The DNA amplificationwas performed at 63∘C for 60min and then terminated byheat at 80∘C for 2min


2000

1000750500

250100

M 1

(a)

M 1 2 3 4 5 6 7 8 9 10 11

2000

1000750500

250100

(b)

Figure 1 Identification of recombinant plasmids by colony PCR (a) M 2 kb ladder marker 1 WT of Bst DNA pol LF gene (b) M 10 kbladder marker 1 WT of Bst DNA pol LF gene 2 LF mutant D540A 3 LF mutant D540E 4 LF mutant G310A 5 LF mutant G310L 6 LFmutant R412A 7 LF mutant R412E 8 LF mutant K416A 9 LF mutant K416D 10 LF mutant G310A-D540E 11 LF mutant G310L-D540E

26 k119888119886119905 Studies of Bst DNA Polymerase In recent yearsvarious studies have applied a rapid and sensitive assay withHPLC to separate and quantify low concentration of dNTPs[18 19] In this study the ion-pair reversed phase HPLCmethod was used to determine the kinetic parameter 119896cat ofBst DNA polymerase by calculating the mass decrement ofdCTP The reaction mixture with high purity containing 4acid-soluble compounds dCTP dGTP dTTP and dATP wasprepared and fixed at 100120583M IMSA assay was performedwith the methods of enzymatic activity at total volume 75120583LThe reaction volume was diluted into 750120583L with ddH2O toprepare the HPLC sample Chromatography was performedwith Symmetry C18 35 120583m (46 times 150mm) column (WatersMilford USA) equipped to a Nova-Pak C18 Sentry GuardColumn with UV detection at 254 nmThemobile phase wasdelivered at a flow rate of 10mLmin with the followinggradient elution program solvent A (10mM tetrabutylam-monium hydroxide 10mM NaH2PO4 and 025 MeOHpH 69)solvent B (56mM tetrabutylammonium hydroxide50mM NaH2PO4 and 30 MeOH adjusted to pH 70) (vv)4060 at 0min 6040 at 30min and 6040 at 60minPolymerization efficiency was measured in triplicate at dCTPconcentration and 119896cat (total dNTPs sec

minus1) was calculated togive turnover numbers of nucleotides incorporated per sec

3 Results

31 Gene Cloning of WT and Mutant Derivatives of Bst DNApol LF To understand the conserved amino acid in dNTPsbinding and polymerization region of Bst DNA pol LF 4amino acid residues mutations of Gly310 Arg412 Lys416 andAsp540 were introduced on the surface of the polymerase cleftby site-directedmutagenesis Bands corresponding to 1758 bpWT of Bst DNA pol LF and 10 genes of site-directed mutantswere detected on 15 (wv) agarose gel by colony PCR ofrecombinant plasmids (Figure 1) demonstrating that WT ofBst DNA pol LF and mutant genes were successfully insertedinto pET21a vectors respectively

32 Expression and Purification of the WT of Bst DNA polLF After OD600 of culture reached mid-logarithm time Ecoli BL21 (DE3) cells containing C-terminal 6x His-tagged

WT of Bst DNA pol fusion protein were induced by adding1mM IPTG and cultured at 37∘C for 6 h Recombinant targetprotein could be observed by Coomassie Blue staining as aprominent band with an apparent molecular mass of 64 kDaafter separation by SDS-PAGE (Figure 2) The recombinantWT of Bst DNA pol LF fusion protein in whole cell lysatewas 154 and 759 of target protein was in soluble fractionThepuritywas determined by the gray scale scanning analysiswith Bio-Rad Quantity One image quantification systemPurification using affinity chromatography is described in theexperimental procedures

33 Expression and Purification of the Derivatives of Bst DNApol LF We constructed eight mutant genes of Bst DNA polLF with individual amino acid substitutions at the tip ofthe ldquothumbrdquo region which is thought to fold over the DNAtemplate The clones were transferred into E coli BL21 (DE3)to express mutant enzymes at reasonably high level followedby protein purification according to the same procedure ofBst DNA pol LF (Figure 3) There was a clear band in eachof the Bst mutant samples at approximately the calculatedmolecular weight of the enzyme (64 kDa) and purity of allsamples prepared using this method was gt95 The puritywas determined as described in Section 32 Interestinglyfrom the SDS-PAGE the molecular weight of the WT ofBst DNA pol LF and the mutant enzymes was a little bitsmaller than that of the commercial Bst 20 DNA polymeraseindicating that the Bst DNA pol LF in this study was notsimilar to market available Bst DNA polymerase

34 IMSA Assay Using the method of visual IMSA to ana-lyze theWT andmutant enzyme activity positive reactions inthe tubes displayed a sky blue color while the negative reac-tions displayed violet color The final result of fluorescencevalues showed that commercial Bst 20 DNA polymerasedisplayed enzyme activityMutantD540E has polymerizationefficiency values identical to WT DNA polymerase whosecurve is two minutes earlier than commercial Bst 20 DNApolymerase Mutant derivatives G310A and G310L showedthree minutes earlier peak than WT DNA polymerase whichdemonstrated Gly310 displaced by Ala or Lys having signifi-cantly increased polymerization efficiency In contrast other


M 1 2 3 4 5 6116

66

45

35

25

18

14

Bst DNA polymerase

Figure 2 SDS-PAGE analysis of the WT of Bst DNA pol LF Mprotein ladder marker shown in kDa on the left sides of panels 1uninduced whole cell sample 2 supernatant fraction of uninducedsample 3 pellet fraction of uninduced sample 4 whole cell sampleafter induction for 6 h 5 supernatant fraction after induction for 6 h6 pellet fraction after induction for 6 h Corresponding position ofBst DNA pol LF was marked by black arrow

M 1 2 3 4 5 6 7 8 9 10 11 12116

66

4535

25

1814

Figure 3 SDS-PAGE analysis of recombinant Bst DNA pol LF andmutant enzymes purified by one-step affinity chromatography Mprotein ladder marker shown in kDa on the left sides of panels 1WT Bst DNA pol LF 2 LF mutant D540A 3 LF mutant D540E4 LF mutant G310A 5 LF mutant G310L 6 LF mutant R412A 7LF mutant R412E 8 LF mutant K416A 9 LF mutant K416D 10 LFmutant G310A-D540E 11 LF mutant G310L-D540E 12 commercialBst 20 DNA polymerase

mutants such as double residues substitution G310A-D540Eand G310L-D540E and the replacement of Asp540 by AlaArg412 by Ala or Glu and Lys416 by Ala or Asp resulted inalmost complete inactivation of the polymerase activity ThemutationD540E does not affect the DNApolymerase activityof the enzyme and presumably there is less distortion of themolecule interaction than the other mutations

35 Kinetic Parameter k119888119886119905 Study of Bst DNA pol LF Thesteady-state kinetic parameter 119896cat (dCTP) was measured inthe presence of saturating concentration of dNTP as shownin Figure 5 The qualities of primers and templates weresufficient in IMSA assay in which standard consumptionof dCTP by HPLC analysis was used to calculate kineticparameter 119896cat (dCTP) of enzymes in positive reaction of visualIMSA assay and sensitivity evaluation assay (WT commercial

Bst DNA pol and mutant derivatives G310L G310A andD540E) The linear relationship between the contents of thedCTP and the chromatographic peak area was investigatedand described by the linear equation119910 = minus17422+2630969119909(1198772 = 0997) Kinetic parameters 119896cat (dCTP) of protein sam-ples in 5 positive reactions in Figure 4 were calculated basedon the linear equation All of these WT and mutants exhibitdifferent 119896cat (dCTP) (S

minus1) from commercial Bst DNA pol andthe results were as follows G310L 0286plusmn 0046 WT 0239plusmn0042 commercial Bst DNApol 0201plusmn0061 G310A 0243plusmn0042 and D540E 0186 plusmn 0039 In conclusion the turnovernumbers for dCTP in IMSA assay catalyzed by Bst DNA poland mutant derivatives followed the order G310L gt G310AgtWT of Bst DNA pol gt D540E gt commercial Bst 20 DNApol Mutations at Gly310 caused an increase in 119896cat implyingthat this amino acid may play an important role in catalyzingDNA polymerization

4 Discussion

HFMD characterized by fever sore throat ulcers in theoral cavity and rashes on the bottoms of the feet and thepalms has been a global common infectious disease causedby a variety of enteric viruses [20] Currently large-scaleoutbreaks of HFMD predominantly characterized by EV71infection a nonenveloped and positive-stranded RNA virushave become a serious public health issue in China thereforeraised the widely public attention and constituted financialburden of government health care system [21] Howeverthere is neither an effective vaccine nor any valid antiviralmedicine for EV71 infection [22] Therefore it is urgent todetect acute infectious HFMD the earlier the diagnosis theeasier the prevention of virus transmission to other childrenand infants to avoid public health emergencies [23] Thedetection limit of LAMP assay is not particularly appropriatefor assessing low viral load specimens in the early phase ofHFMD resulting in a high risk of false-negative diagnosisrate [24] In comparison with the traditional confirmatorydiagnosis as a new type of isothermal amplification tech-nology IMSA assay is a novel real sensitive and promisingmethod based on visual inspection of fluorescence or colorunder natural light changes from negative to positive samples[25] To rapidly test human EV71 visual IMSA assays usingthe highly conserved regions of VP1 gene were developed todevise the corresponding IMSA primers The detect resultsindicated that the normal six-primer combination in theIMSA assay possessed high specificity and sensitivity

This polymerase shares 975 sequence identity withthe previously identified DNA polymerase I of Bacillusstearothermophilus [14] Thermostable DNA polymerase Iused in commercial detection kits has been well protected bypatents leading to relatively high price and restricted appli-cation in China We cloned and recombinantly expressedthe polymerase domain from Geobacillus stearothermophiluswhich is topologically similar to the corresponding domainsof theKlenow fragment consisting of a right hand120572helixwiththree subdomains ldquopalmrdquo ldquofingersrdquo and ldquothumbrdquo to bindduplex DNA [26 27] Slight changes in the conformations of


1 2 3 4 5 6 7 8 9 10 11 12 13

(a)

0

2900

5800

8700

11600

14500

17400

20300

23200

26100

29000

Int

minus+

484218 24 30 36 54 601260

Sample 1 2 3 4 5 6 7 8 9 10 11 12 13

Tt 1830 15001600 1330 1200+++++ minusminusminusminusminusminusminusminus

(b)

Figure 4 Visual IMSA assay and sensitivity evaluation of IMSA assay to test EV71 (a) Visual detection was performed with IMSA assay byaddingHNB dye prior to amplification procedureThe color of sky blue demonstrates positive reactions while the color of violet demonstratesnegative reactionsThe number of the tube indicates IMSA reaction respectively as follows 1 commercial Bst 20 DNA polymerase 2WT ofBstDNApol LF 3 LFmutantD540E 4 LFmutantG310A 5 LFmutantG310L 6 LFmutantD540A 7 LFmutant R412A 8 LFmutant R412E9 LF mutant K416A 10 LF mutant K416D 11 LF mutant G310A-D540E 12 LF mutant G310L-D540E 13 negative control (b) Fluorescencesignals on real-time PCR instrument Fluorescence values and curves were evaluated with Deaou-308C constant temperature fluorescencedetection equipmentThe reaction order in (b) table was arranged the same as tubes number in (a)The sign of ldquo+rdquo indicates positive reactionswhile ldquominusrdquo indicates negative reactions Reactions 1ndash5 were able to amplify VP1 gene to detect EV71 The curves in different colors representdistinct proteins in IMSA reaction Curve in black and ldquoreaction 1rdquo represent commercial Bst 20 DNA polymerase Curve in green andldquoreaction 2rdquo represent WT of Bst DNA pol LF Curve in orange and ldquoreaction 3rdquo represent LF mutant D540E Curve in pink and ldquoreaction 4rdquorepresent LF mutant G310A Curve in red and ldquoreaction 5rdquo represent LF mutant G310L

these domains may affect substrate binding or the polymer-ization efficiency [28] The universally conserved Asp540 inBst DNA pol LF of binding with 31015840-hydroxyl of the primerstrand to form a hydrogen bond is essential for activity of cat-alytic polymerization of dNTPs [29] Asp540 performs a keyrole in catalyzing the formation of the phosphodiester bondThepresence ofMg2+ ionwhich coordinatedwithAsp540 keptBst DNA pol LF staying in the closed conformation In theabsence of metal ion Asp540 forms a hydrogen bond withthe 31015840-hydroxyl which subsequently interactedwith the intro-duced Mg2+ ion and changed the position of sugar puckerat the 31015840-terminus of the primer strand to provide sufficientspace for attacking the 120572-phosphate of the nucleotide [30]The preinsertion site of Bst DNA pol is highly conserved andGly310 at the end of theO helix involved in forming partmotifB in the closed polymerase-DNA-dNTP ternary complexeswhich controlled the template position throughout everycatalytic cycle [9] Gly310 also participated in binding withthe center of a helix-loop-helix motif where the templaterotated 90 degrees along with the template backbone [31]Several mutant derivatives of Bst DNA pol LF based onAsp540 and Gly310 in Bst DNA polymerase were designed andproduced to improve the efficiency of DNA polymerizationThe 119896cat values of positive mutants G310L and G310A were422 and 208 higher than that of the commercial BstDNA polymerase respectively D540E is the other positivemutant in which the 119896cat value is similar to that of commercial

Bst DNA polymerase therefore substitutions at the activesite Gly310 and Asp540 were made simultaneously to improveefficiency of DNA polymerization It was disappointing thatmost of the substitutions were with significant loss of DNApolymerase activityThedramatic changes inmutantsG310A-D540E and G310L-D540E highlight that Gly310 and Asp540have a profound effect on stabilizing the catalytic site ofBst DNA polymerase In this situation one mutation maybe sufficient to alter the substrate polymerization but moresite-directed mutagenesis is detrimental to the stability oractivity of Bst DNA polymerase by destroying hydrogenbonds to water molecules or other highly conserved residues(Arg325 Glu368 Gln507 and His539) of polymerase activesite that participates in the network of hydrogen bondsand electrostatic interaction [29] All of these residues havesignificant effect on substrate binding or catalysis when theywere mutated Adopting optimal reaction conditions theefficiency and sensitivity of Bst DNA polymerases in IMSAassay were evaluated with the artificial template The WT orcommercial Bst DNApolymerase was chosen as parallel testsModified Bst DNA polymerase (G310L) displayed higherefficiency ofDNAamplification than theWTand commercialsamples

In summary the modified Bst DNA polymeraseemployed in IMSA technology on the one hand breaksapplication restrictions of the patent in commercial kit inChina on the other hand it improved the understanding


29

36 30

88 3

364

373

5

95

30 12

560

13

737

16

583

22

609

26

531

43

175

0

10

20

30

40

50(m

AU)

10 20 30 40 50 60 700(min)

(a)

30

49 3

367

363

0

77

59 98

63 13

236

17

447

24

227

28

559

49

860

0

5

10

15

20

25

30

(mAU

)

10 20 30 40 50 60 700(min)

(b)

30

82 3

312

367

4

96

79 12

804

17

059

23

319

27

475

45

548

05

1015202530

(mAU

)

10 20 30 40 50 60 700(min)

(c)

30

74 3

370

364

4

98

51 13

126

17

454

24

014

28

255

47

859

70

104

05

1015202530

(mAU

)

10 20 30 40 50 60 700(min)

(d)

Figure 5 HPLC analysis of polymerization efficiency of Bst DNA polymerases in IMSA assay (a) Negative control retention time of dCTPis 16583min and the peak area is 245942 (b) the LF mutant G310L retention time of dCTP is 17447min and the peak area is 178162 (c)Bst DNA pol WT retention time of dCTP is 17059min and the peak area is 184069 (d) commercialized Bst 20 DNA polymerase retentiontime of dCTP is 17454min and the peak area is 194152

of the three-dimensional structure of Bst DNA polymeraseand the mechanism of DNA amplification This study hasestablished a fast simple accurate and sensitive diagnosticmethod for rapidly detecting HFMD associated with EV71which provides an efficient way for quick identificationespecially for the primary diagnostics setting in rural andunderdeveloped areas

Competing Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

This work was supported by the Project of Produc-tion and Research Collaborative Innovation of Guangzhou(201508020110) the National Natural Science Foundationof China (21306055) the International Cooperation Pro-gramme ofDepartment of Science andTechnology ofGuang-dong Province (2015A050502016) the Natural Science Foun-dation of Guangdong Province China (2014A030313261) thePhD Programs Foundation of Ministry of Education ofChina (20130172120041) Project of Natural Science Foun-dation of Guangdong Province (2016A030313457) and the

Fundamental Research Funds for the Central Universities(2015ZM161)

References

[1] T Notomi H Okayama H Masubuchi et al ldquoLoop-mediatedisothermal amplification of DNArdquo Nucleic Acids Research vol28 no 12 article e63 2000

[2] I P Oscorbin U A Boyarskikh and M L Filipenko ldquoLargefragment of DNA polymerase I from Geobacillus sp 777cloning and comparison with DNA polymerases I in practicalapplicationsrdquo Molecular Biotechnology vol 57 no 10 pp 947ndash959 2015

[3] M Imai A Ninomiya H Minekawa et al ldquoDevelopment ofH5-RT-LAMP (loop-mediated isothermal amplification) sys-tem for rapid diagnosis of H5 avian influenza virus infectionrdquoVaccine vol 24 no 44-46 pp 6679ndash6682 2006

[4] J P Dukes D P King and S Alexandersen ldquoNovel reversetranscription loop-mediated isothermal amplification for rapiddetection of foot-and-mouth disease virusrdquoArchives of Virologyvol 151 no 6 pp 1093ndash1106 2006

[5] M Parida K Horioke H Ishida et al ldquoRapid detection anddifferentiation of dengue virus serotypes by a real-time reversetranscription-loop-mediated isothermal amplification assayrdquoJournal of Clinical Microbiology vol 43 no 6 pp 2895ndash29032005


[6] K A Curtis D L Rudolph and S M Owen ldquoRapid detectionof HIV-1 by reverse-transcription loop-mediated isothermalamplification (RT-LAMP)rdquo Journal of Virological Methods vol151 no 2 pp 264ndash270 2008

[7] Y Kurosaki A Takada H Ebihara et al ldquoRapid and simpledetection of Ebola virus by reverse transcription-loop-mediatedisothermal amplificationrdquo Journal of Virological Methods vol141 no 1 pp 78ndash83 2007

[8] X Ding K Nie L Shi et al ldquoImproved detection limit in rapiddetection of human enterovirus 71 and coxsackievirus A16 by anovel reverse transcription-isothermal multiple-self-matching-initiated amplification assayrdquo Journal of Clinical Microbiologyvol 52 no 6 pp 1862ndash1870 2014

[9] M Delarue O Poch N Tordo D Moras and P ArgosldquoAn attempt to unify the structure of polymerasesrdquo ProteinEngineering vol 3 no 6 pp 461ndash467 1990

[10] A Hasan A A Abedi and S Arjunan ldquoMolecular characteri-zation of thermostable DNA polymerase of Bacillus stearother-mophilus spp isolated from soil in Bangalore Indiardquo EuropeanJournal of Experimental Biology vol 4 no 4 pp 67ndash72 2014

[11] J M Aliotta J J Pelletier J L Ware L S Moran J S Bennerand H Kong ldquoThermostable Bst DNA polymerase I lacks a31015840 rarr 51015840 proofreading exonuclease activityrdquo Genetic AnalysisBiomolecular Engineering vol 12 no 5-6 pp 185ndash195 1996

[12] T A Steitz ldquoDNA polymerases structural diversity and com-mon mechanismsrdquo Journal of Biological Chemistry vol 274 no25 pp 17395ndash17398 1999

[13] S-M Phang C-Y Teo E Lo and V W Thi Wong ldquoCloningand complete sequence of the DNA polymerase-encoding gene(BstpolI) and characterisation of the Klenow-like fragmentfrom Bacillus stearothermophilusrdquo Gene vol 163 no 1 pp 65ndash68 1995

[14] F C Lawyer S Stoffel R K Saiki et al ldquoHigh-level expres-sion purification and enzymatic characterization of full-lengthThermus aquaticus DNA polymerase and a truncated formdeficient in 51015840 to 31015840 exonuclease activityrdquo Genome Research vol2 pp 275ndash287 1993

[15] Y XuO Potapova A E LeschzinerND FGrindley andCMJoyce ldquoContacts between the 51015840 nuclease of DNA Polymerase Iand Its DNA substraterdquo Journal of Biological Chemistry vol 276no 32 pp 30167ndash30177 2001

[16] M G Riggs S Tudor M Sivaram and S H McDonoughldquoConstruction of single amino acid substitution mutants ofcloned Bacillus stearothermophilus DNA polymerase I whichlack 5

1015840

rarr 31015840

exonuclease activityrdquo Biochimica et BiophysicaActa vol 1307 no 2 pp 178ndash186 1996

[17] F Van Den Ent and J Lowe ldquoRF cloning a restriction-freemethod for inserting target genes into plasmidsrdquo Journal ofBiochemical and Biophysical Methods vol 67 no 1 pp 67ndash742006

[18] P Chen Z Liu S Liu et al ldquoA LC-MSMS method forthe analysis of intracellular nucleoside triphosphate levelsrdquoPharmaceutical Research vol 26 no 6 pp 1504ndash1515 2009

[19] E Kinai H Gatanaga Y Kikuchi S Oka and S KatoldquoUltrasensitive method to quantify intracellular zidovudinemono- di- and triphosphate concentrations in peripheral bloodmononuclear cells by liquid chromatography-tandem massspectrometryrdquo Journal of Mass Spectrometry vol 50 no 6 pp783ndash791 2015

[20] Y Jing W X Huang H W Zeng et al ldquoHand-foot-and-mouthdiseaserdquo in Diagnostic Imaging of Emerging Infectious DiseasesSpringer Amsterdam The Netherlands 2016

[21] T P Van Boeckel S Takahashi Q Liao et al ldquoHand foot andmouth disease in China critical community size and spatialvaccination strategiesrdquo Scientific Reports vol 6 Article ID25248 2016

[22] L-J Xu T Jiang F-J Zhang et al ldquoGlobal transcriptomic anal-ysis of human neuroblastoma cells in response to enterovirustype 71 infectionrdquo PLoS ONE vol 8 no 7 Article ID e659482013

[23] E Dias and M Dias ldquoRecurring hand foot mouth disease in achildrdquo Annals of Tropical Medicine and Public Health vol 5 no1 pp 40ndash41 2012

[24] X Wang J-P Zhu Q Zhang et al ldquoDetection of enterovirus71 using reverse transcription loop-mediated isothermal ampli-fication (RT-LAMP)rdquo The Journal of Virological Methods vol179 no 2 pp 330ndash334 2012

[25] X Ding W Wu Q Y Zhu T Zhang W Jin and Y MuldquoMixed-dye-based label-free and sensitive dual fluorescence forthe product detection of nucleic acid isothermal multiple-self-matching-initiated amplificationrdquo Analytical Chemistry vol 87no 20 pp 10306ndash10314 2015

[26] H Klenow and I Henningsen ldquoSelective elimination of theexonuclease activity of the deoxyribonucleic acid polymerasefrom Escherichia coliB by limited proteolysisrdquo Proceedings of theNational Academy of Sciences of theUnited States of America vol65 no 1 pp 168ndash175 1970

[27] D L Ollis P Brick R Hamlin N G Xuong and T A SteitzldquoStructure of large fragment ofEscherichia coliDNApolymeraseI complexedwith dTMPrdquoNature vol 313 no 6005 pp 762ndash7661985

[28] A H Polesky T A Steitz N D F Grindley and C M JoyceldquoIdentification of residues critical for the polymerase activity ofthe Klenow fragment of DNA polymerase I from Escherichiacolirdquo The Journal of Biological Chemistry vol 265 no 24 pp14579ndash14591 1990

[29] J R Kiefer C Mao C J Hansen et al ldquoCrystal structure of athermostable Bacillus DNA polymerase I large fragment at 21A resolutionrdquo Structure vol 5 no 1 pp 95ndash108 1997

[30] C A Brautigam and T A Steitz ldquoStructural and functionalinsights provided by crystal structures of DNA polymerasesand their substrate complexesrdquo Current Opinion in StructuralBiology vol 8 no 1 pp 54ndash63 1998

[31] Y Zhao D Jeruzalmi I Moarefi L Leighton R Laskenand J Kuriyan ldquoCrystal structure of an archaebacterial DNApolymeraserdquo Structure vol 7 no 10 pp 1189ndash1199 1999

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of


Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology


Molecular Biology International

GenomicsInternational Journal of


The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014


BioinformaticsAdvances in

Marine BiologyJournal of



Signal TransductionJournal of


BioMed Research International

Evolutionary BiologyInternational Journal of



Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014


Genetics Research International


Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational



Enzyme Research



Microbiology


Since Kornberg discovered and studied DNA polymeraseI in detail from Escherichia coli in 1958 [9] various DNApolymerases such as DNA polymerases 120572 and 120573 and RNApolymerase have been isolated and characterized from bothprokaryotic and eukaryotic organisms [10 11] The indis-pensable Bst DNA polymerase I in the isothermal ampli-fication was widely applied and played a key role in clin-ical rapid detection of virus infections Similar to otherDNA polymerase I Bst DNA polymerase from Bacillusstearothermophilus consisted of three independent domainswhich perform biological function individually (I) 51015840-31015840exonuclease activity (II) 51015840-31015840 DNA polymerase activity and(III) 31015840-51015840 exonuclease activity [12] The domains II andIII are located in the C-terminus of Bst DNA polymerasedesignated as the large fragment (LF) [13] The full-lengthof Bst DNA polymerase I gene consisted of 2634-base-pairwhile the truncated LF gene devoid of the 51015840-terminal 876-base-pair could be translated into a predicated 671 kD DNApolymerase with C-terminal poly (His)6 which lacks theinherent 51015840-31015840 proofreading exonuclease activity and wasextremely efficient in polymerizing dNTPs in PCR [14]Since Bst DNA polymerase meets all requirements for highsensitivity polymerization activity and thermal stability itis extensively applied in isothermal amplification procedureof rapid detection However Bst DNA polymerase is onlyavailable popular in some international companies as NewEnglandBiolabs at high priceTherefore it is urgent to designproduce and purify a new and efficient Bst DNA polymeraseto satisfy growing demand of less developed area In thisstudy we cloned the gene of Bst DNA polymerase I froma new strain (Geobacillus stearothermophilus GIM1543) andmake various site-directed substitutions of four conservedresidues in the polymerase active site which is important inbinding the DNA primer terminus and dNTP to catalyze thepolymerase reaction [15 16] Three mutants showed higherpolymerization activity than the commercial Bst DNA poly-merase and consequently would lay the roots for promotingits wide application

2 Materials and Methods

21 Strains Plasmids Enzymes and Reagents Geobacillusstearothermophilus was purchased from Microbial CultureCollection Center of Guangdong Institute of MicrobiologyChina Competent E coli cells DH5120572 and BL21 (DE3) werepurchased fromTIANGENBiotech (Beijing China) PlasmidpET21a was preserved by our own laboratory Restrictionendonucleases Nde I and Xho I were purchased from Ther-moFisher Scientific Co Ltd (Shanghai China) Reagentsrelated to Bst DNA pol LF gene cloning and site-directedmutagenesis were purchased from TaKaRa BiotechnologyCo Ltd (Dalian China) The HisTrapFF column from GEHealthcare (Piscataway NJ USA) was used for proteinspurification The chemicals used for HPLC were obtainedfrom Sigma-Aldrich Chemical (St Louis USA) All chemi-cals used in this study were of analytical grade

22 Construction of Expression Vector Genome DNA fromGeobacillus stearothermophilus was prepared with a genome

extraction kit (Sangon Shanghai China) according to man-ufacturerrsquos handbook The coding region of LF in BstDNA polymerase (Bst pol LF) using PrimeSTAR HS DNApolymerase was amplified with the following primers theforward primer was 51015840-CTGTTCCATATGGAAGGCGAA-AAGCCGCTCGCC-31015840 and the reverse primer was 51015840-CCGCTCGAGTTTGGCGTCGTACCACGTC-31015840 Restric-tion sites Nde I and Xho I used for subsequent amplificationare shown in bold and underline PCR was implementedunder the following condition initial heating at 95∘C for5min 30 cycles of 95∘C for 30 s 56∘C for 1min and 72∘Cfor 10min followed by an extension step at 72∘C for 5minThe PCR product of 1758 bp DNA fragment was separated by15 agarose gel electrophoresis and purified using a DNA gelextraction kit (Sangon Shanghai) Purified PCR product wasdigested with Nde I and Xho I and ligated into pET21a vector(Novagen Madison) yielding the plasmid pET21a-Bst-LF-H6with one poly (His)6 tag at C-terminus and transformed intoE coli DH5120572 cells Recombinant DH5120572 cells were spreadon LuriandashBertani (LB) agar plates containing 50 ng120583L ofkanamycin and incubated at 37∘C overnight The positiveclones were confirmed by colony PCR and DNA sequenceanalysis The sequence similarity of Bst DNA pol LF waschecked by the BLAST program

23 Mutagenesis Experiment Multiple protein sequencesincluding WT target protein sequence were aligned and ana-lyzed Several amino acid residues were selected as mutationsites shown in Table 1 Using theWT gene of Bst DNA pol LFas template the Restriction Free (RF) cloning PCR method[17] was applied to construct the mutated genes with primerpairs shown in Table 1 The Bst DNA pol LF proteins wereexpressed with C-terminal 6x His-tag promoting proteinspurification All purified proteins (WT and mutants) and thecommercial Bst 20 DNA polymerase were evaluated by SDS-PAGE

24 Expression and Purification of the WT and MutantDerivatives of Bst DNA pol LF Plasmid pET21a-Bst-LF-H6 was transformed into E coli BL21 (DE3) (NovagenMadison WI USA)The recombinant bacteria were inducedto express recombinant LF-Bst-H6 by adding isopropyl-120573-D-thiogalactopyranoside (IPTG) at a final concentration of1mM to a culture with OD600 of approximately 06ndash08 andincubating at 37∘C for 6 h Cell culture (1 L) was harvestedby centrifugation at 5000timesg for 30min The cell pelletswere resuspended in 40mL 1x PBS buffer (50mM Tris-HCland 300mM NaCl) and lysed with 450 sonication pulses(400W 3 s with a 5 s interval) cooled in ice water bath Thesuspension was centrifuged (11000timesg at 4∘C for 30min) andpassed through a 022 120583m filter followed by applying to a 5mL HiTrapFF chelating HP column The target protein Bst-LF-H6 was purified using the standard nickel affinity chro-matography procedure and washed with 5 column volumeseach of 50mM 100mM 150mM and 200mM imidazole incolumn buffer (20mM Tris-HCl 500mM NaCl and 20glycerol pH 80) followed by gel-filtration on Bio-Gel P-6(Bio-Rad Laboratories Inc) according to the manufacturerrsquos





















2000

1000750500

250100

M 1

(a)

M 1 2 3 4 5 6 7 8 9 10 11

2000

1000750500

250100

(b)




3 Results







M 1 2 3 4 5 6116

66

45

35

25

18

14

Bst DNA polymerase


M 1 2 3 4 5 6 7 8 9 10 11 12116

66

4535

25

1814






4 Discussion




1 2 3 4 5 6 7 8 9 10 11 12 13

(a)

0

2900

5800

8700

11600

14500

17400

20300

23200

26100

29000

Int

minus+

484218 24 30 36 54 601260

Sample 1 2 3 4 5 6 7 8 9 10 11 12 13


(b)






29

36 30

88 3

364

373

5

95

30 12

560

13

737

16

583

22

609

26

531

43

175

0

10

20

30

40

50(m

AU)

10 20 30 40 50 60 700(min)

(a)

30

49 3

367

363

0

77

59 98

63 13

236

17

447

24

227

28

559

49

860

0

5

10

15

20

25

30

(mAU

)

10 20 30 40 50 60 700(min)

(b)

30

82 3

312

367

4

96

79 12

804

17

059

23

319

27

475

45

548

05

1015202530

(mAU

)

10 20 30 40 50 60 700(min)

(c)

30

74 3

370

364

4

98

51 13

126

17

454

24

014

28

255

47

859

70

104

05

1015202530

(mAU

)

10 20 30 40 50 60 700(min)

(d)



Competing Interests


Acknowledgments



References


















1015840

rarr 31015840
























Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology





















2000

1000750500

250100

M 1

(a)

M 1 2 3 4 5 6 7 8 9 10 11

2000

1000750500

250100

(b)




3 Results







M 1 2 3 4 5 6116

66

45

35

25

18

14

Bst DNA polymerase


M 1 2 3 4 5 6 7 8 9 10 11 12116

66

4535

25

1814






4 Discussion




1 2 3 4 5 6 7 8 9 10 11 12 13

(a)

0

2900

5800

8700

11600

14500

17400

20300

23200

26100

29000

Int

minus+

484218 24 30 36 54 601260

Sample 1 2 3 4 5 6 7 8 9 10 11 12 13


(b)






29

36 30

88 3

364

373

5

95

30 12

560

13

737

16

583

22

609

26

531

43

175

0

10

20

30

40

50(m

AU)

10 20 30 40 50 60 700(min)

(a)

30

49 3

367

363

0

77

59 98

63 13

236

17

447

24

227

28

559

49

860

0

5

10

15

20

25

30

(mAU

)

10 20 30 40 50 60 700(min)

(b)

30

82 3

312

367

4

96

79 12

804

17

059

23

319

27

475

45

548

05

1015202530

(mAU

)

10 20 30 40 50 60 700(min)

(c)

30

74 3

370

364

4

98

51 13

126

17

454

24

014

28

255

47

859

70

104

05

1015202530

(mAU

)

10 20 30 40 50 60 700(min)

(d)



Competing Interests


Acknowledgments



References


















1015840

rarr 31015840
























Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


M 1 2 3 4 5 6116

66

45

35

25

18

14

Bst DNA polymerase


M 1 2 3 4 5 6 7 8 9 10 11 12116

66

4535

25

1814






4 Discussion




1 2 3 4 5 6 7 8 9 10 11 12 13

(a)

0

2900

5800

8700

11600

14500

17400

20300

23200

26100

29000

Int

minus+

484218 24 30 36 54 601260

Sample 1 2 3 4 5 6 7 8 9 10 11 12 13


(b)






29

36 30

88 3

364

373

5

95

30 12

560

13

737

16

583

22

609

26

531

43

175

0

10

20

30

40

50(m

AU)

10 20 30 40 50 60 700(min)

(a)

30

49 3

367

363

0

77

59 98

63 13

236

17

447

24

227

28

559

49

860

0

5

10

15

20

25

30

(mAU

)

10 20 30 40 50 60 700(min)

(b)

30

82 3

312

367

4

96

79 12

804

17

059

23

319

27

475

45

548

05

1015202530

(mAU

)

10 20 30 40 50 60 700(min)

(c)

30

74 3

370

364

4

98

51 13

126

17

454

24

014

28

255

47

859

70

104

05

1015202530

(mAU

)

10 20 30 40 50 60 700(min)

(d)



Competing Interests


Acknowledgments



References


















1015840

rarr 31015840
























Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


1 2 3 4 5 6 7 8 9 10 11 12 13

(a)

0

2900

5800

8700

11600

14500

17400

20300

23200

26100

29000

Int

minus+

484218 24 30 36 54 601260

Sample 1 2 3 4 5 6 7 8 9 10 11 12 13


(b)






29

36 30

88 3

364

373

5

95

30 12

560

13

737

16

583

22

609

26

531

43

175

0

10

20

30

40

50(m

AU)

10 20 30 40 50 60 700(min)

(a)

30

49 3

367

363

0

77

59 98

63 13

236

17

447

24

227

28

559

49

860

0

5

10

15

20

25

30

(mAU

)

10 20 30 40 50 60 700(min)

(b)

30

82 3

312

367

4

96

79 12

804

17

059

23

319

27

475

45

548

05

1015202530

(mAU

)

10 20 30 40 50 60 700(min)

(c)

30

74 3

370

364

4

98

51 13

126

17

454

24

014

28

255

47

859

70

104

05

1015202530

(mAU

)

10 20 30 40 50 60 700(min)

(d)



Competing Interests


Acknowledgments



References


















1015840

rarr 31015840
























Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


29

36 30

88 3

364

373

5

95

30 12

560

13

737

16

583

22

609

26

531

43

175

0

10

20

30

40

50(m

AU)

10 20 30 40 50 60 700(min)

(a)

30

49 3

367

363

0

77

59 98

63 13

236

17

447

24

227

28

559

49

860

0

5

10

15

20

25

30

(mAU

)

10 20 30 40 50 60 700(min)

(b)

30

82 3

312

367

4

96

79 12

804

17

059

23

319

27

475

45

548

05

1015202530

(mAU

)

10 20 30 40 50 60 700(min)

(c)

30

74 3

370

364

4

98

51 13

126

17

454

24

014

28

255

47

859

70

104

05

1015202530

(mAU

)

10 20 30 40 50 60 700(min)

(d)



Competing Interests


Acknowledgments



References


















1015840

rarr 31015840
























Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology













1015840

rarr 31015840
























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 enhancement of polymerase activity of the...

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