Cloning and high-level expression of Thermus thermophilus RecA in E. coli: purification and novel use in HBV diagnostics

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h ttp : / / w w w . b j m i c r o b i o l . c o m . b r /

Biotechnology

and

Industrial

Microbiology

Cloning

and

high-level

expression

of

Thermus

thermophilus

RecA

in

E. coli:

puriﬁcation

and

novel

use

in

HBV

diagnostics

Sudarson

Sundarrajan,

Sneha

Rao,

Sriram

Padmanabhan

∗

CancyteTechnologiesPvt.Ltd.,RangadoreMemorialHospital,SriShankaraResearchCenter,Shankarapuram,Bangalore,India

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received4October2017

Accepted13March2018

Availableonline12April2018

AssociateEditor:FlávioDaFonseca

Keywords: RecA HepatitisBvirus DNAvirus Thermusthermophilus

a

b

s

t

r

a

c

t

WestudiedtheroleofThermusthermophilusRecombinaseA(RecA)inenhancingthePCR

signalsofDNAvirusessuchasHepatitisBvirus(HBV).TheRecAgeneofathermophilic

eubacterialstrain,T.thermophilus,wasclonedandhyperexpressedinEscherichiacoli.The

recombinantRecAproteinwaspuriﬁedusingasingleheattreatmentstepwithouttheuseof

anychromatographysteps,andthepuriﬁedprotein(>95%)wasfoundtobeactive.The

puri-ﬁedRecAcouldenhancethepolymerasechainreaction(PCR)signalsofHBVandimprove

thedetectionlimitoftheHBVdiagnosisbyrealtimePCR.TheyieldofrecombinantRecA

was∼35mg/L,thehighestyieldreportedforarecombinantRecAtodate.RecAcanbe

suc-cessfullyemployedtoenhancedetectionsensitivityforthediagnosisofDNAvirusessuch

asHBV,andthismethodologycouldbeparticularlyusefulforclinicalsampleswithHBV

viralloadsoflessthan10IU/mL,whichisinterestingandnovel.

anopenaccessarticleundertheCCBY-NC-NDlicense(http://creativecommons.org/

licenses/by-nc-nd/4.0/).

Introduction

TherecombinaseA(RecA)protein(accessionno.AAK15321.1)

from the thermophilic bacterium Thermus thermophilusis a

thermostableenzymethatplaysimportantrolesin

homolo-gousrecombinationandDNArepair.1_DNA_{recombination}_in

prokaryotesisachievedbycatalyzingthepairingof

homol-ogous DNA sequences,2,3 _and _DNA _repair _is _achieved _by

∗ _{Corresponding}_author_at:_R_&_D,_Cancyte_Technologies_Pvt._Ltd,_Sri_Shankara_Research_Centre,_Rangadore_Memorial_Hospital,_1st_Cross,

Shankarapuram,Bangalore560004,India.

E-mails:sriram.padmanabhan@cancyte.com,padsriram@gmail.com(S.Padmanabhan).

protection of the damaged DNA ends.4,5 _This _protein _has

theactivitiesofsingle-strandedDNAdependentATPase,DNA

annealing, andexchangingofstrands betweentwo

recom-bining DNA double helices, similar to Escherichia coli RecA

(accessionno.AFU91764.1)protein,buttheoptimal

tempera-tureforthestrandexchangeofRecAisbetween50and60◦C.6

RecA-like proteins have been sequenced from over 60

differentprokaryoticspecies7,8 _and_it _hydrolyzes_ATP_when

boundtoDNA,anditsATPaseactivityrateisproportionalto

https://doi.org/10.1016/j.bjm.2018.03.007

(2)

theamountofRecAboundtotheDNA9–12_with_the_fact_that

theATPaseactivityisusedtomeasureitsbindingefﬁciency

toDNA.13_It_has_been_shown_that_the_RecA_protein_catalyzes

invitroDNAstrandexchangereactions14_with_peak_efﬁciency

atapproximately60◦C,6_a_temperature_that_is_fairly_close_to

theTmrequiredforthermaldenaturationofdsDNA.15

NumerousapplicationsofRecAarereported.Theseinclude

identiﬁcationofdifferentBurkholderia cepaciagenomovars,16

sequence-speciﬁc ligation of DNA using the E. coli RecA

protein,17 _maintaining _the _integrity _of _chloroplast _DNA

moleculesinArabidopsis,18_{auto-cleavage}_of_other_serine

pro-teasesandrepressorsofthebacteriophagelyticcycle,19–22_and

itsroleinswarmingmotilityinE.coli.23

Shigemorietal.24_have_used_RecA_in_stimulating_pairing

betweencompletelymatchedprimersandtargetsequences

and effectivelyeliminating non-speciﬁcPCRproducts. This

speciﬁcityenabledthemtoamplifythesequences between

anysitesofinterestwithoutneedingtoconsidertheoptimal

primingsitesforeachPCR.Similarly,FukuiandKuramitsu25

have recently utilized MutS, a thermostable

mismatch-recognizingproteinfromT.thermophilus,alongwithRecAto

reducenon-speciﬁcampliﬁcationsinPCRs.

Hence,werealizedtheimmensepotentialofRecAin

PCR-baseddiagnosticsandattemptedtogenerateRecAproteinby

asimplepuriﬁcationmethodthatwouldinturnmakethePCR

baseddetectionofvirusesmoresensitiveandcost-effective.

Material

and

methods

Reagents

Allchemicalswereofanalyticalgrade.Oligonucleotides,used

inthisstudy,werefromBioServeTechnologiesPvt.Ltd,

Hyder-abad,India. Bradfordproteinassayreagentsand IPTGwere

obtained from Amresco, USA. Standard RecA pure protein

was procured from NEB, USA, while ATP was purchased

fromThermoFisherScientiﬁc,USA.Proteinmolecularweight

markers 11–180kDa and 11–97kDa were obtained from

G-Biosciences, USA and Aristogene Life Sciences, Bangalore,

Indiarespectively.

CloningandconstructionofRecAexpressionconstruct

ThesyntheticgeneofT.thermophilusRecAwascustom

syn-thesizedfromIntegratedDNATechnologies,Inc.(IDT),USA,

andclonedintothepUC57vector.Thenucleotidesequence

oftheT.thermophilusRecAgenewasadjustedforcodonbias

withoutalteringanyoftheaminoacids.TheRecAgenewas

excisedfromthepUC57-RecAplasmidasaNdeI/HindIII

frag-mentandclonedintothepET26bvectorintosimilarrestriction

sites.TherecombinantswerescreenedbyPCRusingT7

pro-moterspeciﬁcprimers,forward5TAATACGACTCACTATAGGG

3 andreverse5 GCTAGTTATTGCTCAGCGG3,andthe

posi-tivecloneswerefurtherconﬁrmedbyrestrictionanalysisand

DNAsequencing.ThepET26b-RecAplasmidwasdesignatedas

pCAN008.

TheplasmidDNAofpCAN008wasintroducedintoER2566

competent cells (NEB,USA) for expression ofrecombinant

RecA.TheprotocolwasessentiallyasdescribedbyPauletal.26

Brieﬂy, theinduction ofRecA waseffected bythe addition

of1mMIPTGwhencellsinLBmedium(containing20␮g/mL

kanamycin)reachedanOD600valuebetween0.5and0.8.The

inductionproceededfor4hbyincubatingthecultureat37◦C,

andthentheinducedcellswerepelletedat7670×gfor10min.

Theharvestedcellswereresuspendedin25mMTris.Cl,pH

7.5andsonicatedinasonicator(OmniSonicRuptor400,GA,

USA)at50%amplitudeforoneminon/offfor10minincold.

Thesonicatedlysatewasspunat9000×gfor20minat4◦C

andthesupernatantobtainedwasusedforpuriﬁcationofthe

RecAexpressedprotein.

RecApuriﬁcationbyheattreatment

RecA is known to be thermostable,27 _and _we _utilized _this

propertytopurifytherecombinantRecAprotein.Different

vol-umesofcrudelysateofRecAwereheatedat75◦Cfor30min,

after which the sampleswere centrifuged at30,670×g for

10min,andthesupernatantswereanalyzedon12%SDS–PAGE

forRecAproteinfollowedbysilverstainingasdescribedby

Nesterenkoetal.28 _Protein_{concentrations}_were_determined

byBradford’smethod29_to_quantify_the_total_yield_of_RecA_per

literofinducedcultureunderthedescribedexperimental

con-ditions.

ATPhydrolysisassay

ATPase activity was determined using a QuantiChrom

ATPase/GTPaseAssayKitDATG-200(EnzyChrom,USA).

Phos-phatestandardsweremadebytakingvaryingconcentrations

ofphosphate(0–50␮M)innucleasefreewaterasper

manu-facturer’sinstructions.Equalamountsofproteinsofin-house

puriﬁedRecAandcommercialRecA(NewEnglandBiolabs,UK)

wereusedfordetectingtheATPaseactivity.Afterincubating

theRecAproteinswithATP,200␮Lofthesuppliedreagentwas

added,andcontentswereincubatedfor30minatroom

tem-perature. TheOD ofthesamples,reﬂecting freephosphate

releaseduetoATPhydrolysisbyRecA,wasreadatOD620nmin

anELISAplatereader(TecanGroupLtd,Männedorf,

Switzer-land).

Humanleucocyteantigen(HLA)PCR

HumangDNAisthesourceofthetemplateforthe

ampliﬁ-cationofHLAgenes.30_The_different_protocols_for_HLA_typing

reportedrequireuseofgDNAbetween5and30ng/␮L31 _and

DNA concentrations lower than 2ng/␮L have been found

to compromise genotyping results severely.32 _Since _RecA

is known to prevent false priming, we hypothesized that

sampleswith lower concentrationsofgenomic DNA might

display highergene ampliﬁcationsin presenceofRecA.To

validatethis hypothesis,wecarriedout PCRsofHLAgenes

from humangDNA inpresence and absence of RecA.The

primers for ampliﬁcation of HLA class I genes, namely,

HLA-A-exon 2and exon 3, HLA-B-exon 2and exon 3 and

HLA-C-exon2and3,wereasdescribedbyItohetal.,33_while

theprimersforHLAclassIIgenessuchasDPB1,DQB1 and

DRB1,targetingexon2,wereaccordingtoLangeetal.30_The

PCRreactionwascarriedoutin25␮Lreactionvolumeandthe

(3)

2.5mMmagnesiumchloride;0.2mMdNTPs;5%DMSO;0.2%

Tween-20;0.04%BSA;0.4␮MHLAprimers;and1unitofTaq

DNA polymerase (ThermoFisher Scientiﬁc, USA)with 40ng

ofgDNA.PCRconditionswereasfollows:Initialdenaturation

of95◦Cfor5min,followedby35cyclesof95◦C,30s;51.1◦C,

30s;and72◦C,45s.Theﬁnalextensionstepwasperformed

at72◦Cfor10min,andsuitablealiquotswereloadedona2%

agarosegelforvisualization.

SamplecollectionandHIV,HBVandHCVPCR

Patients who visited Kuppam Medical College, Andhra

Pradesh,IndiaforviraltestingofHIV,HBVandHCVduringthe

periodofMarch2016toMarch2017servedasthestudy

mate-rial.BloodsampleswerecollectedinEDTAcoatedtubesand

plasmawascollectedbycentrifugingthesamplesat2000×g

for10min.Theplasmasampleswerestoredat−20◦Cuntil

fur-theruse.Insomecases,serumwascollectedfromthepatients.

PlasmaorserumsampleswereanalyzedforHIV,HBVand

HCVbyroutinediagnostictests(RDT)aspermanufacturer’s

instructions.ThetestkitsemployedincludedtheHIV-1

TRI-DOTkit,HEPACARDtestkitandHCVTRI-DOTtestkits.Viral

positivesampleswerehandledwithadequateprecautionsin

theBSL-IIfacilityofCancyteTechnologiesPvt.Ltd,Bangalore,

India.IVDcertiﬁedNucleospinDxviruskit(Macherey-Nagel,

GmbH)wasusedtoisolateviralnucleicacidsfollowing

man-ufacturer’sinstructions.

TominimizetheriskofcontaminationofPCRamplicons,

andtoavoidtransferofDNAfromonesampletotheother,

inourlaboratory,thepreandpost-polymerasechainreaction

(PCR)productswerekeptphysicallyseparated,andall

biolog-icalsampleswereprocessedseparatelyinadedicatedBSL-II

biosafetyhood.TheDNA-freeconsumablesandnegative

con-trolswereusedateverystageofthePCRanalysis.

TheHIVprimerswere designedtoamplify thegaggene

ofthe HIV(accession number KY713247.1), while the HBV

andtheHCVprimersampliﬁedtheSgene(accession

num-ber MF967563.1) of HBV and 5 UTR (accession number

MG436884.1)regionofHCVrespectively.AllthePCRswere

car-riedoutin25␮LreactionvolumesandthePCRproductswere

analyzedon2%agarosegelelectrophoresisstainedwith

ethid-iumbromideforvisualization,unlessmentionedotherwise.

The PCR mixture comprised 1× Superscript III

RT/Platinum® Taq Mix buffer, HIV forward primer (5

ATCAAGCAGCCATGCAAAT3)andreverseprimer(5

TACTAG-TAGTTCCTGCTATGTC3)or HCVforward (5 GAAAGC GTC

TAGCCATGGCG3)andreverseprimer(5ACGCCCAAATGG

CCGGGCATAGA3),and0.5␮LofSuperScriptIIIenzyme.PCR

wasperformedinaProﬂexPCRmachine(AppliedBiosystems,

USA).PCRconditionswereasfollows:onecycleof50◦Cfor

30mintoconvertRNAtocDNA,followedbyasinglestepof

denaturationat95◦Cfor5minand40cyclesof95◦Cfor30s,

55◦Cfor30s,and72◦Cfor1minfollowedbyaﬁnalextension

at72◦Cfor10min.

PCR for HBV detection in clinical samples was carried

out in25␮Lreactionvolumes. ThePCRmixturecomprised

of 1× ammonium sulfate buffer, pH 8.3; 2.5mM

magne-siumchloride;0.2mMdNTPs;5%DMSO;primers0.4␮M(FP

5 CCCCCACTGGCTGGGGCTTGGT 3 and RP5

AGGACGTC-CCGCGCAGGATC 3); and 1unit of Taq DNA polymerase

(ThermoFisherScientiﬁc,USA).PCRstepsincludedaninitial

denaturationof95◦C,10min,followedby40cyclesof95◦C,

30s;60◦C,30s;and72◦C,45sandaﬁnalextensionstepat

72◦Cfor10min.

For carryingoutPCRswithRecAandATP, ATPwasused

ataﬁnalconcentrationof0.3mMalongwith0.25␮gofeither

in-housepuriﬁedRecAorcommerciallyavailableRecA.

HBVviralload

Brieﬂy,thereactionwascarriedoutinaﬁnalvolumeof25␮L

containing1×ThermoScientiﬁcMaximaSYBRGreenmaster

mix,andforward(FP5CCCCCACTGGCTGGGGCTTGGT3and

RP5AGGACGTCCCGCGCAGGATC3)primerswereaddedtoa

ﬁnalconcentrationof4pmolperreaction.Atotalof0.3mM

ATPand0.25␮gofRecAwereaddedtooneset,andsuitable

sampleswithoutRecAservedasnegativecontrols.

TheqPCRwasperformedinaQiagenqPCRmachine

(Qia-gen,Germany),andthe conditionsforqPCR were45 cycles

ofPCRampliﬁcation,eachcomprising95◦Cfor5min,95◦C

for30s,60◦Cfor30s,and72◦Cfor30s.Datacollectionwas

performed at72◦C in each cycle. Following the

ampliﬁca-tion, amelting curve analysiswas performedto verify the

authenticityoftheampliﬁedproductsbycheckingtheir

spe-ciﬁcmelting temperatures(Tm).Experimentswere donein

duplicates.

Therecombinantplasmid containingthe SgeneofHBV

(named pTZ57R/T-HBV) was conﬁrmed by DNA

sequenc-ing (data not shown) and used to generate a standard

curve. The concentration (ng/␮L) of the puriﬁed stock of

plasmid DNA was measured by a NanoDrop 1000

spec-trophotometer(ThermoFisherScientiﬁc),andplasmidcopy

number was calculated using the DS DNA copy calculator

usingtheformula:pTZ57R/Tcopies/␮L=(plasmid

concentra-tion[ng/␮L]×6.022×1023)/(nucleotidelength×1×109×650),

asdescribedbyStaroscik.34_The_plasmid_stock_solution_was

dilutedwithDNase/RNase-freedistilledwater,10-foldserial

dilutionscontaining2.5×105_–250_copies/_␮L_were_made,_and

smallaliquotswerestoredat−20◦_C_for_future_use_in

generat-ingstandardcurves.TheCtvaluesweretestedforstatistical

signiﬁcancebyStudent’st-test.

Results

Cloningandexpressionstudies

Fig.1showstheplasmidmapofpET26b-RecA.Theclonesof

pET26b-RecAwereconﬁrmedbyDNAsequencingand

restric-tionanalysis.Fig.S1showsthesilverstainedgelofwholecell

lysatesoftwoclonesofpET26b-RecA.TherecombinantRecA

proteinwasrestrictedtothesolublefractionofthecelland

constituted>50%ofthetotalsolubleproteinasjudgedby

den-sitometryscanningandoftheexpectedsizeof∼39kDa.The

studiesontheexpressionofRecAfrompET26b-RecAclonein

ER2566cellsusingdifferentconcentrationsofIPTGindicated

thattheamountofRecAexpressionincreasedwithincreased

timeofinductionasexpected(Fig.S2)andthattheexpression

ofRecAcouldbeinducedevenwith0.125mMIPTGeffectively

(4)

Dra l

Pvu ll

Cla l

Kan _{lac Z} Hind lll

Alw l Ori T7 pro pCAN 008/ Rec A 6309 bp Rec A Gene (1064) Nde l

Fig.1–PlasmidmapofthepET26b-RecAconstruct (pCAN008). 1 M 2 3 4 75 63 48 35 25

Fig.2–SilverstainedSDS–PAGEgelofthepuriﬁed

in-houseRecAandcommercialRecAproteins.Notethatthe purityoftheRecAis>95%asjudgedbythesilverstained gel.Lane1:commercialRecA(0.25␮g);lane2:in-house purifiedRecA(0.2␮g);lane3:in-housepurifiedRecA (0.5␮g);lane4:in-housepurifiedRecA(1.0␮g).M: pre-stainedproteinMWmarker(11–180kDa).

PuriﬁcationandtotalyieldofRecAusingheattreatment

TheRecAexpressedfromtheclonepET26b-RecA(#5)afterheat

treatmentwasfoundtobehomogenousandwas>95%pure

asjudgedbysilverstaining.ItisevidentfromFig.S4thatheat

treatmentwassufﬁcienttoachievepureRecA.Thetotalyield

ofpureRecAperliterofLBwasfoundtobe35mg/Landits

M 9 8 7 6 5 4 3 2 1

A

B

Fig.3–HLA-PCRofClassIandClassIIantigensbyPCR. PanelAshowsPCRwithoutRecA,andpanelBshowsHLA PCRswithRecA.Lane1:HLA-A-Exon2(478bp);lane2: HLA-A-Exon3(327bp);lane3:HLA-B-Exon2(469bp);lane 4:HLA-B-Exon3(371bp);lane5:HLA-C-Exon2(489bp); lane6:HLA-C-Exon3(500bp);lane7:DPB1,Exon3(368bp); lane8:DQB1,Exon3(350bp);lane9:DRB1Exon3(351bp); lane10:DNAmolecularweightmarker(100bpladder).

puritymatchedthepurityofthecommerciallyavailableRecA

protein(Fig.2).

ATPhydrolysisassay

Thein-housepuriﬁedRecA proteinshowed ATPaseactivity

units of3.0×105_U/_␮L,_while_the _commercial _RecA _showed

activityof2.5×105_U/␮L._This_result_indicated_that_the

puriﬁ-cationmethodologyfollowedforthein-houseRecAprotein

doesnotcompromisethefunctionoftheRecAprotein.

HLA-PCR

ItisclearfromFig.3thatRecAampliﬁestheHLAgenesof

inter-estevenwhenthegDNAusedwasaslowas5ngperreaction,

whilewithoutRecA,therewasnegligibleampliﬁcationofall

thegenesundersimilarexperimentalconditions.

HIV,HBVandHCVPCR

Fig.4showsthePCRampliﬁcationofHIV/HBVandHCVtarget

genesusingthreeELISApositiveclinicalsamples.Itisevident

fromFig.4thatRecAimprovedthePCRsignalintensitiesofthe

HBVsamples(Fig.4,lanes13,14and15incomparisontolanes

4,5and6),whilethePCRsignalsoftargetgenesofRNAviruses

(5)

1 2 3 4 5 6 M 7 8 9

10 11 12 13 14 15 M 16 17 18

A

B

Fig.4–PCRproductsofthreeHIV,HBVandHCVsamples withandwithoutRecA.PanelAshowsPCRwithoutRecA, whilepanelBshowsPCRwithRecA.Lanes1,2,3,10,11 and12areHIVsamples;lanes4,5,6,13,14,and15are HBVsamples;lanes7,8,9,16,17,and18areHCVsamples. RT-PCRwasperformedfortheHIVandHCVsamplesas describedintheM&Msection,whileforHBV,regularPCR wasperformedaspertheprotocoldescribedintheM&M section.

therewasmarkedreductioninlevelsofnon-speciﬁc

ampli-ﬁcationswhenRecAwasincludedinthePCRreactionmix.

SinceHBVisaDNAvirus,itispossibletospeculatethatRecA

enhancestheannealingoftheHBVspeciﬁcprimerstothe

tar-getSgeneoftheHBV,andduetothereportedroleofRecAin

minimizingfalsepriming,theintensityofthePCRsignalsfor

theHBVsamplesappearhigherwithRecAincomparisonto

thePCRofthesamesampleswithoutRecA.

HBVviralload

SinceourdatashowedthatthePCRsignalsofHBVwashigher

inpresenceofRecAincomparisontothatachievedwithout

RecA,wewantedtoexamineifRecAwouldalsohaveaneffect

ontheHBVviralloadvaluesintheHBVpositiveplasma

sam-ples.FromFig.5A,itisclearthatthesensitivityoftheHBV

detectionisenhancedandtheoverallHBVviralloadvalues

were higher (by 50–98%) forall the samplestested in this

studywhenRecAwasincludedinthePCRreactionmixture

incomparisontotheHBVviralloadvaluesobtainedforthe

samesampleswithoutRecA(Table1).Fig.5Bshowsthe

stan-dardcurvepreparedusingplasmidpTZ57R/T-HBVfrom250to

2.5×105_copies/mL.

Discussion

TheT7promoterisknowntobethestrongestpromoterknown

forthe prokaryotic expressionofproteins35 _and _in

experi-mentalE.colicultures,consistentlyhigherexpressionlevelsof

theproteinhavebeenobservedwiththekanamycinmarker

than inculturesgrownwiththecommonlyused antibiotic

ampicillin36_with_the_target_protein_being_almost_50%_of_total

A

B

1 2 3 4 5 6 7 M +ve -ve 1 2 3 4 5 6 7 M +ve -ve 38 36 34 32 30 28 26 24 22 20 18 16 14 CT 102 ₁₀3 ₁₀4 ₁₀5 ₁₀6 ₁₀7 Concentration

Cycling A.Green (Page1): R=0.99928 Rˆ2=0.99855 M=-3.944 B=42.653 Efficiency=0.79

Fig.5–(A)AgarosegeldepictingPCRofHBVsamples. PanelAshowsPCRwithoutRecA,whilepanelBshowsPCR withRecAforthesamesamples.Lanes1,2,3,4,5,6and7 showPCRampliconsfromELISApositiveHBVsamples.+ve denotespositivecontrolPCRwithpTZ57R/T-HBVplasmid and−vedenotesPCRwithnotemplatecontrol.(B) RepresentativestandardcurveoftheSYBRGreen-1qPCR assayforHBV.Theplotsweregeneratedusinga10-fold dilutionseriesrangingfrom250to2.5×105_copies/mL_of

plasmidDNAstandard(pTZ57R/T).Duplicatereactionsfor eachstandarddilutionwereused.Thestandardcurvewas generatedbyplottingthelog10copynumberofstarting

plasmidquantityonthex-axisagainstthecorresponding thresholdcycle(Ct)valueonthey-axis.

cellular proteininsomecases.37 _It_is _possible_to_speculate

thatfortheabovereasons,weachievedhighlevelsofRecA

expressiondescribedinthisstudy.

OnecouldobserveasmallfractionofRecAproteininduced

evenunderuninducedconditions(Fig.S4,lane1).Expression

ofRecAunderuninducedconditionscouldbeattributedtothe

leakyexpressionoftheT7promoterthatisseencommonly

inE.colicellsandcanbeeasilycontrolledusingplysE

plas-mid,which expressesT7lysozyme,aninhibitorofT7RNA

polymerase.38 _Also, _one _could _see _appreciable _amounts _of

RecA expressedevenwiththelowestconcentrationofIPTG

(0.125mM) which supports the recent report of high level

expressionofaleptospiralproteininE.coliusingsuchlow

concentrationsofIPTG.39

A number of protocols have been reported for

puriﬁ-cation of wild-type RecA protein from E. coli or Thermus

aquaticus. These methods include salt precipitation

(6)

Table1–HepatitisBvirus(HBV)viralloadvaluesofenzyme-linkedimmunesorbentassay(ELISA)positiveHBVsamples withandwithoutRecA.

Sample Ctvaluewithout

RecA

WithoutRecA (VL/mL)

Cyclethreshold(Ct) valuewithRecA*

WithRecA (VL/mL) %increaseinVL withRecAin comparisontoVL withoutRecA Sample#1 38.515 2.00E+02 35.605 5.99E+03 96.66 Sample#2 41.155 2.00E+02 37.285 4.60E+03 95.65 Sample#3 15.41 6.20E+08 13.65 4.52E+09 86.29 Sample#4 29.58 1.70E+05 26.75 3.59E+05 52.59 Sample#5 34.185 1.20E+04 31.035 2.95E+04 59.28 Sample#6 38.605 1.00E+02 34.36 2.55E+04 99.61 Sample#7 41.175 2.00E+02 37.67 4.10E+03 95.12

∗ _The_Ct_values_were_found_to_be_{statistically}_signiﬁcant_(p_<_0.000001)_by_Student’s_‘t’_test.

exchangecolumn,25_{precipitation}_using_spermidine,40

puriﬁ-cationusing chitinafﬁnity column,41 _monoclonal _antibody

afﬁnitycolumns,42_and _{phosphocellulose}_matrix.43 _Many_of

theseprotocolsrequiremultiplestepsforpuriﬁcationandare

notverycost-effective.Inanefforttodevelopasimpleanda

cost-effectiveprotocolforRecApuriﬁcation,wetook

advan-tageoftheheatstablepropertyofRecAandobtainedhighly

pureRecA preparationsbya simpleheat treatment ofthe

crudelysatewithoutthe useofanychromatographic steps

andsuchamethodologyforthepuriﬁcationofRecAishitherto

unreported.

Asigniﬁcantamountofendonucleasecontaminationfrom

thehostendAgenehasbeenreportedinRecAprotein

prepa-rationsbySingletonetal.,41_which_was_removed_by_rigorous

washingoftheinduced cellswithdetergents.Theprotocol

describedinthisarticleutilizesanendA1negativeE.colihost,

ER2566,forRecAexpression,andhence,ourpuriﬁedRecAis

virtuallyfreeofanyendonucleasecontamination,whichisa

hugeadvantageforitsuseinPCR-baseddiagnostics.

TheRecAgeneofT.thermophilusencodesa36kDa

polypep-tidewhoseaminoacidsequenceshows61%identitywiththat

oftheE.coliRecAproteinandT.thermophilusRecAisreported

tobethermostableincomparisontoE.coliRecAprotein27_with

similarfunctions.TheyieldofrecombinantRecA(fromE.coli

orT.thermophilus)fromvariousliteraturereportsisvariable

andnotfeasibleforcommercialscaleoperations.Whilethe

protocoldescribedbyGrifﬁthandShores40_yield_only₁₀_mg

ofRecAproteinperliter,theprotocoldevelopedbySingleton

etal.41_showed_a_yield_of₂₈_mg_of_RecA/liter_of_E._coli_culture.

Inthisstudy,wehaveachieved35mgofpureRecAperliter

ofE.coliculture,whichisthehighestreportofRecAyieldtill

date.Additionally,thecostof1mgofcommercialRecAis$575

andweareabletoproducealmostUS$20,125worthRecAfrom

everyliterofE.coliculture,whichisinterestinganduseful.

Lactosecanalsobeusedasaninducerduringthetransition

fromtheexponentialtothestationaryphaseforprotein

induc-tionforgenesclonedunderT7promoter.Sincelactosealso

actsascarbonsource,theuseoflactoseforinductionwould

proveusefulforincreasingtheRecAexpressionpercell.44

ATPhydrolysisservesasameansofreleasingRecA

promot-ersfromthe5endoftheﬁlamentonceDNAstrandexchange

occurs,thus helpsrecyclethe RecA protein.45 _{Additionally,}

ithasbeenshownbyYuand Egelman46 _that_the_RecA

ﬁla-mentisinactivewhenthecompressedﬁlamentisformedin

theabsenceofanucleotidecofactorsuchasATP,eitherasa

self-polymer oronasingle-strandedDNAmolecule.Hence,

wehypothesizedthatATPhydrolysisisacrucialstepforthe

activityofRecA,andhence,theATPaseactivityofRecAcan

serveasameasureformonitoringRecAstrandexchange

activ-ity.WeobservedthattheATPaseactivityofthein-houseRecA

andcommercialRecApreparationsweresubstantiallysimilar.

ThisdataindicatesthatthepuriﬁedRecAdoesnothavethe

propertyofaggregation,sincetheaggregationstateofRecA

proteinisknowntoaffectitsATPaseactivity.47

Shigemorietal.24_have_performed_RT-PCR_for_their_target

geneusingaSYBRpremixExTaqkitwithandwithoutRecA

andobservedanearly30%increaseinPCRﬂuorescencesignal

intensitywithRecAcomparedtothatwithoutRecA.Our

obser-vationsof∼50to96%increaseintheﬂuorescenceintensity

oftheHBV PCRswithRecAsupportsthereported

observa-tions.Inaddition,weshowlowerCtvaluesinHBVqPCRwith

RecA,which isanewobservation.Also,the limitof

detec-tion(LOD)oftheHBVviralloadmethoddevelopedusingSYBR

greendyeappearstobe5viruscopies/reactionwhichequates

to40IU/mL.ThisLODvalueisvery closetotheLODvalue

oftheAbbottHBVviralloadkit(utilizingTaqManchemistry)

of15IU/mLforaprocedureprocessing0.2mLofthestarting

sample.

Anassaywithalower limitofdetection of103IU/mLof

HBVisreportedtobesufﬁcienttomonitorandmanagethe

patient,48_but_in_some_instances,_a_more_sensitive_assay_with

alowerlimitofdetectionontheorderof101_IU/mL_is

recom-mendedtoensuredetectionoftheemergenceofresistanceas

earlyaspossible.Hence,ourobservationsofenhancementof

the limitofHBV viralloaddetectionbyalmost 1log copies

can beextrapolated toimproving theqPCR HBV LODfrom

200copies/mLto20copies/mLor40IU/mLto4IU/mL,which

ishithertounreported.

In this paper, wehave described a real-time PCR assay

forHBVDNAquantiﬁcationwithSYBRGreenchemistry.The

primersusedforqPCRtargetsthesurfaceantigenofHBVand

havebeendesignedafteraligningnucleotidesequencesofall

theHBVgenotypesavailableintheNCBIdatabasesothatone

isassuredofpositivePCRsignalforallHBVserotypesusing

thesesetofprimers.

Viral load set pointhas long been usedas aprognostic

markerofdiseaseprogressionand monitoringofvirusload

(7)

HBVstrains.Also,anundetectableorlowviralloaddoesnot

necessarilyguaranteethatapatientwillnotexperienceliver

damage;hencemethodsthatenhancethesensitivityofthe

viralloadmethodwouldhaveimmensevalueinclinical

set-ting and further drug treatments. In the light of this, our

observationofRecAenhancingtheLODoftheviralload

esti-mationmethodisnovelandinteresting.

We are presently focusing on generating novel RecA

fusionstoexaminewhethertheheatstabilityofRecAcanbe

impartedtoheterologousgenesandwhethersuch

heterolo-gousRecAfusionproteinscouldbepuriﬁedbyheattreatment

asdescribedinthisarticle.

Conﬂicts

of

interest

Theauthorsdeclarenoconﬂictsofinterest.

Acknowledgements

TheauthorswishtothankDr.C.Nagaraj,Prof.ofMicrobiology,

PESIMSR,Kuppam,India,forthesupplyofclinicalsamplesof

HIV,HBVandHCVforourstudies.Theauthorsalsowishto

thankDr.Sridhar,K.N.andDr.GauthamNadigfortheir

con-stantsupportandencouragement.CancyteTechnologiesPvt.

Ltd.isfullysupportedbySriSringeriSharadaPeetam,India.

Appendix

A. Supplementary

data

Supplementarydataassociatedwiththisarticlecanbefound,

intheonlineversion,atdoi:10.1016/j.bjm.2018.03.007.

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