ANALYTICALBIOCHEMISTRY

Analytical Biochemistry 329 (2004) 11–20

www.elsevier.com/locate/yabio

Toward pyrosequencing on surface-attached genetic materialby use of DNA-binding luciferase fusion proteins

Maria Ehn, Nader Nourizad, Kristina Bergström, Afshin Ahmadian, Pål Nyrén, Joakim Lundeberg, and Sophia Hober¤

Department of Biotechnology, Royal Institute of Technology, S-106 91 Stockholm, Sweden

Received 24 October 2003Available online 30 April 2004

Abstract

Mutation detection and single-nucleotide polymorphism genotyping require screening of large samples of materials and thereforethe importance of high-throughput DNA analysis techniques is signiWcant. Pyrosequencing is a four-enzyme bioluminometric DNAsequencing technology based on the sequencing-by-synthesis principle. Currently, the technique is limited to simultaneous analysis of96 or 384 samples. Earlier, attempts to increase the sample capacity were made using micromachined Wlter chamber arrays whereparallel analyses of nanoliter samples could be monitored in real time. We have developed a strategy for speciWc immobilization ofthe light-producing enzyme luciferase to the DNA template within a reaction chamber. By this approach, luciferase is geneticallyfused to a DNA-binding protein (Klenow polymerase or Escherichia coli single-stranded DNA-binding (SSB) protein) and to a puri-Wcation handle (Zbasic). The proteins are produced in E. coli and puriWed using cation and anion exchange chromatography withremoval of Zbasic. The produced proteins have been analyzed using an assay for complete primer extension of DNA templates immo-bilized on magnetic beads detected by pyrosequencing chemistry. Results from these experiments show that the proteins bind selec-tively to the immobilized DNA and that their enzymatic domains were active. Zbasic-SSB-luciferase produced the highest signal inthis assay and was further exploited as enzymatic reagent for DNA sequencing. 2004 Elsevier Inc. All rights reserved.

Keywords: Luciferase; SSB; Klenow; Pyrosequencing; Sample capacity

The innovation of DNA sequencing technology pyrophosphate (PPi)1 is released. The PPi is converted to

enabled reading the genetic code of all living organismsand is thus a great landmark for all research in the Weldof molecular biology. Most de novo DNA sequencingtechniques are based upon DNA synthesis using Xuores-cently labeled nucleotide analogues followed by electro-phoretic separation of labeled fragments and detection[1]. However, several alternative techniques are currentlyavailable and their further development is of greatimportance.

Pyrosequencing technology is a sequencing methodusing real-time bioluminometric detection of DNA syn-thesis [2]. When the DNA polymerase incorporatesnucleotides into the growing DNA chain, inorganic

¤ Corresponding author. Fax: +46-8-553-784-81.E-mail address: sophia@biotech.kth.se (S. Hober).

0003-2697/$ - see front matter 2004 Elsevier Inc. All rights reserved.doi:10.1016/j.ab.2004.02.005

adenosine 5�-triphosphate (ATP) by ATP sulfurylase [3]and Wnally the ATP serves as substrate for the light-producing enzyme luciferase [4]. Thus, if a nucleotideis incorporated into the DNA template, a charge-cou-pled device (CCD) camera detects a light signal emittedby luciferase. The bases are consequently added to the

1 Abbreviations used: PPi inorganic pyrophosphate; CCD, charge-coupled device; ATP, adenosine 50-triphosphate; SNP, single-nucleotidepolymorphism; PCR, polymerase chain reaction; IPTG, isopropyl-�-D-thiogalactopyranoside; OD, optical density; AU, absorbance units;CVs, column volumes; SDS–PAGE, sodium dodecyl sulfate polyacryl-amide gel electrophoresis; BSA, bovine serum albumin; Tris, tris(hy-droxymetyl)aminomethane; EDTA, ethylenediamintetraacetic acid;CIE, cation exchange chromatography; AIE, anion exchange chroma-tography; SSB, single-stranded DNA-binding; TSB, tryptic soy broth;YE, yeast extract; DTT, dithiothreitol; APS, adenosine 50-phosphosul-phate.

12 M. Ehn et al. / Analytical Biochemistry 329 (2004) 11–20

reaction mixture one at a time iteratively in a cyclic man-ner. To remove unincorporated nucleotides and ATPbetween each base dispension, an enzyme that degradesthose substrates (apyrase) [5] is included in the system.

The pyrosequencing technology has been success-fully used in large studies of single-nucleotide poly-morphism (SNP) genotyping and mutation detection(exempliWed by [6] and [7], respectively) in a 96-wellplate format. However, for the technique to be compet-itive with regard to sample throughput, an increase incapacity would be beneWcial. Systems containing sev-eral parallel Wlter chambers have been produced [8,9].Furthermore, SNP analysis by allele-speciWc extensionmonitored by light production according to thepyrosequencing principle has been performed in a sin-gle 12.5-nl Wlter chamber [9,10]. In these experiments,the DNA sample was immobilized on streptavidin-coated beads captured in the Wlter chambers and thereaction components were Xown through the Wlterchamber. Since all reagents used were soluble, theycould not be captured within the Wlter chamber and themajority of the light was therefore detected in the outletof the chamber.

Here, we have developed a novel strategy based ongene fusion technology for docking of the light-produc-ing enzyme to the DNA template. Two fusion proteinscontaining a thermostable variant of luciferase fused toa DNA-binding protein domain and a highly chargedZbasic tag for selective and eYcient protein recoveryunder mild conditions have been constructed [11]. ThepuriWcation handle can be selectively removed by site-speciWc cleavage using the viral 3C-protease [12–14]. Athermostable variant of the enzyme of Photinus pyralis,containing the four amino acid substitutions E354 ! K,T214 ! A, I232 ! A, and F295 ! L [15] was used for thefusion proteins. The aims of using a thermostable vari-ant of the enzyme were to increase expression levels andto allow for higher Xexibility in the design of experimen-tal protocols. Two diVerent approaches for acquiringDNA aYnity of the luciferase fusion were used. In onecase, the DNA-binding of Escherichia coli single-stranded DNA-binding (SSB) protein was exploited[16,17]. Moreover, this protein has proven to improveperformance in pyrosequencing technology [18] and tostimulate the eVect of various enzymes [19–21]. KlenowDNA polymerase [22] was used in the second strategy,taking advantage of its interaction with the DNA tem-plate and its polymerase activity.

This article describes the production, downstreamprocessing, and characterization of the two fusion pro-teins. Their abilities of binding speciWcally to DNA tem-plates with maintained enzyme activities of the luciferaseand polymerase domains are shown. Finally, the applica-bility of Zbasic-SSB-luciferase as a template-immobilizedenzymatic reagent in pyrosequencing analysis is demon-strated.

Materials and methods

Bacterial strains and plasmids

E. coli strains RRI�M15 [23] and BL21(DE3) (Nova-gen, Madison, WI) were used as bacterial host duringcloning and protein production, respectively. The plas-mids used as templates for PCR ampliWcation werepPWLTx4 (luciferase) [15] and pTrp-SSB (SSB) [24] whilethose used for cloning were pT7ZbasicII, pT7ZbasicII-Klenow [14], and pRIT28-Klenow [25]. The vectorpPWLTx4 was a kind gift from Dr. Tisi and containedfour point mutations (T214 ! A, I232 ! A, F295 ! L, andE354 ! K) [15] compared to the wild-type luciferase fromP. pyralis.

Oligonucleotides

The oligonucleotides were purchased from Interac-tiva GmbH (Interactiva Biotech, Ulm, Germany) andMWG-BIOTECH AG (MWG-BIOTECH AG, Ebers-berg, Germany). Primers used for PCR ampliWcation ofthe thermostable luciferase were EHMA 8, 50-AAGACAGGATCCATGGAAGACGCCAAAAACATAAAG-30

and EHMA 9, 50-TTGGCAAGCTTACATTTTACACTTTGGACTTTCCGC-30. By use of the EHMA 9, anamino acid change from L to V was obtained at position550 in the luciferase domain. Primers used for PCRampliWcation of SSB were EHMA 21, 50-AAAAAAAAAAAAAAGAATTCCATGGCCAGCAGAGGCGTAAAC-30 and EHMA 22, 50-ACTTTGATGATATTCCGTTCGGATCCGTCGACAAAAAAAAA-30. In thesolid phase DNA analysis experiments, two biotinylated90-mer oligonucleotides, OLIGOII and OLIGOIII [18],were immobilized on streptavidin-coated magneticbeads and the MALO3b primer [18] was annealed to the90-mer oligonucleotide.

Construction of expression vectors

All recombinant DNA technology was performedaccording to standard procedures [26].

ZbasicII-Klenow-luciferase. The thermostable variantof the luciferase gene was PCR ampliWed from thepPWLTx4 vector using primers EHMA 8 and EHMA9, which introduced a BamHI site upstream of theluciferase gene and a HindIII site downstream. Lucifer-ase was subcloned into the pRIT28-Klenow vectordownstream of Klenow DNA polymerase usingBamHI and HindIII. After DNA sequencing of theluciferase gene, a DNA fragment containing the C-ter-minal part of the Klenow and the whole luciferase wascleaved out from the plasmid using SacI and HindIII.The gene fragment was cloned into the pT7ZbasicII-Klenow vector and the Wnal construct was called pT7-ZbasicII-Klenow-luciferase. This plasmid harbors

M. Ehn et al. / Analytical Biochemistry 329 (2004) 11–20 13

kanamycin resistance, and protein production is undercontrol of the T7 promoter.

ZbasicII-SSB-luciferase. The SSB gene was PCRampliWed from pTrpSSB using the EHMA 21 andEHMA 22 primers. The primers introduced an EcoRIsite upstream of the SSB gene and SalI downstream. Byuse of these enzymes, the ampliWed gene fragment wassubcloned into the pT7ZbasicII vector C-terminal of theZbasic gene. The thermostable variant of the luciferasewas cleaved out of the pT7-ZbasicII-Klenow-luciferaseplasmid using BamHI and HindIII and ligated intopT7ZbasicII-SSB. The resulting plasmid was calledpT7ZbasicII-SSB-luciferase and is identical to the pT7-ZbasicII-Klenow-luciferase vector except that the Klenowgene is substituted for the SSB fragment.

Production of proteins

A colony containing pT7ZbasicII-Klenow-luciferase orpT7ZbasicII-SSB-luciferase was grown overnight at 30 °Cin a shake Xask containing 50 ml of tryptic soy broth(TSB) + yeast extract (YE) (30 g/L tryptic soy broth;Merck KgaA, Darmstadt, Germany; 5 g/L yeast extract)supplemented by kanamycin (50 mg/L). The followingmorning, cultivations were inoculated with 10 ml of theovernight culture to 500 ml fresh TSB + YE with kana-mycin (50 mg/L) in 5-L shake Xasks. The cells weregrown at 30 °C until OD600 nm reached approximately1.0. Protein production was induced by addition of iso-propyl-�-D-thiogalactopyranoside (IPTG) to a Wnalconcentration of 1 mM. Cells were harvested by centrifu-gation after protein production had taken place for 3.5 h(pT7ZbasicII-Klenow-luciferase) or 5.5 h (pT7ZbasicII-SSB-luciferase).

Downstream protein processing

Cell harvest. Cells were harvested by centrifugation atapproximately 4000g for 10 min and cell pellets corre-sponding to 500 ml culture were resuspended in 50 ml of50 mM Tris–HCl, pH 7.5. The cell suspensions were kepton ice. Proteins were released by sonication (Vibracell,Sonics & Materials, Danbury, CT) and cell debris wereseparated by centrifugation at 20,000g for 20 min. Thesoluble fractions were Wltered through a 0.45-�m Wlterand the conductivity was adjusted to 20 mS/cm by addi-tion of 5 M NaCl. Samples were taken out for sodiumdodecyl sulfate-polyacrylamide gel electrophoresis(SDS–PAGE).

Cation exchange chromatography. A XK16 columncontaining 24 ml S-Sepharose FF cation exchange resin(Amersham Biosciences, Uppsala, Sweden) was preequil-ibrated with 5 column volumes (CVs) of 50 mM Tris–HCl, pH 7.5, with 200 mM NaCl (buVer A). The Wltratewas loaded onto the column using the liquid chromatog-raphy system ÄKTA (Amersham Biosciences) at a linear

Xow rate of 60 cm/h. Unbound material was washed outfrom the column with approximately 5 CVs of buVer Aat a linear Xow rate of 60 cm/h followed by 180 cm/h untilthe absorbance at 280 nm was below 50 mAU. Zbasic-Klenow-luciferase was eluted using a linear NaCl gradi-ent of 200–1000 mM/20 CV and Zbasic-SSB-luciferasewas eluted by stepwise increased 0–500–750–1000 mMsalt concentration (Figs. 1A and 2A). Eluted protein wascollected in 7-ml fractions and screened for luciferaseactivity. The purity of luciferase-containing fractionswas analyzed using SDS–PAGE and relevant fractionswere pooled.

Fig. 1. Downstream processing of SSB-luciferase. (A) Chromatogramfrom cation exchange puriWcation of Zbasic-SSB-luciferase (B) Chro-matogram from anion exchange puriWcation of SSB-luciferase afterremoval of the Zbasic puriWcation handle. (C) SDS–PAGE analysis ofthe downstream processing of SSB-luciferase. M* is a high-molecular-weight marker containing protein standards of 53.0, 76.0, 116.0, and170.0/220.0 kDa. M is a low-molecular-weight marker containing pro-tein standards of 14.4, 20.1, 30.0, 45.0, 66.0, and 94.0 kDa. The analyzedfractions, given from the left, are 1, material loaded on the cationexchange column; 2, cation exchange-puriWed material; 3, Protease-cleaved material; and 4, anion exchange-puriWed material.

14 M. Ehn et al. / Analytical Biochemistry 329 (2004) 11–20

Site-speciWc 3C-protease cleavage. Prior to proteasecleavage, the protein samples were diluted with MilliQwater until the salt concentration was below 300 mMNaCl. The proteins to be cleaved were mixed with Zbasic-3C at a molar ratios 1:1 (Zbasic-Klenow-luciferase) and1:5 (Zbasic-SSB-luciferase). Cleavage was allowed to takeplace in room temperature overnight in presence of5 mM DTT.

Anion exchange chromatography. The cleavage mix-tures were diluted with 50 mM Tris–HCl, pH 8.5, andMilliQ water to conductivity below 5 mS/cm and pH

Fig. 2. Downstream processing of Klenow-luciferase. (A) Chromato-gram from cation exchange puriWcation of Zbasic-Klenow-luciferase.(B) Chromatogram from anion exchange puriWcation of Klenow-lucif-erase after removal of the Zbasic puriWcation handle. (C) SDS–PAGEanalysis of the downstream processing of Klenow-luciferase. M* is ahigh-molecular-weight marker containing protein standards of 53.0,76.0, 116.0, and 170.0/220.0 kDa, respectively. M is a low-molecular-weight marker containing protein standards of 14.4, 20.1, 30.0, 45.0,66.0, and 94.0 kDa, respectively. The analyzed fractions, given from theleft, are 1, material loaded on the cation exchange column; 2, cationexchange-puriWed material; 3, protease-cleaved material; and 4, anionexchange-puriWed material.

about 8. The puriWcation was performed at a linear Xowrate of 0.1 cm/h using the liquid chromatography systemÄKTA (Amersham Biosciences) and a 1-ml Resource Qanion exchange column (Amersham Biosciences) pre-equilibrated with 10 CVs of 50 mM Tris–HCl, pH 8.0(buVer A). After sample loading, unbound material waswashed out of the column using 10 CVs of buVer A. Elu-tion of SSB-luciferase was performed using a linear gra-dient of 0–250 mM NaCl/50 CVs followed by a secondlinear gradient of 250–1000 mM NaCl/16 CVs. In thepuriWcation of Klenow-luciferase, an extra wash with 10CVs of buVer A with 50 mM NaCl preceded the elutionconsisting of linear gradients of 50–150 mM NaCl/40CVs and 150–1000 mM NaCl/18 CVs. Eluted proteinfractions were analyzed by luciferase activity assay andSDS–PAGE and relevant fractions were pooled. To fur-ther conWrm the yield of this puriWcation step, the chro-matograms from CIE and AIE puriWcations of pooledfractions were integrated and compared for the twofusion proteins.

Protein characterization: screening between puriWcationsteps

Luciferase activity. Fractions from ion exchangechromatography were screened for luciferase activityusing a luminometric assay monitored on a LKB 1251tube luminometer. Ten microliters of the samples wereadded to a mixture containing 0.1 M Tris–acetate (pH7.75), 2 mM EDTA, 10 mM Mg–acetate, 0.1% bovineserum albumin (BSA), 1 mM DTT, 0.4 mg/ml polyvinyl-pyrrolidone, and 100 �g/ml D-luciferin. The reaction wasinitiated by addition of 1 �l 10�M ATP and the lumines-cence was measured on the luminometer connected to apotentiometric recorder.

Protein purity. Fractions from diVerent puriWcationsteps showing luciferase activity were analyzed by SDS–PAGE using a Phast system (Amersham Biosciences).The gels were run under reducing conditions and stainedwith Coomassie brilliant blue R-350 according to thesupplier's recommendations. Gels used were of 4–15%(Klenow-luciferase) or 10–15% (SSB-luciferase) gradienttypes (Amersham Biosciences). Prior to Coomassiestaining, the separated proteins were thermoblotted to anitrocellulose Wlter by raising the temperature in thePhast system to 70 °C for 20 min. For detection of lucif-erase, the membrane was incubated with rabbit poly-clonal anti-luciferase (Sigma Chemical, St. Louis, MO)diluted 1:1000 in 50 mM Tris–HCl, pH 7.5, 0.15 M NaCl.For detection of Zbasic, it was incubated with rabbit poly-clonal anti-protein A (Sigma Chemical) diluted 1:5000 in50 mM Tris–HCl, pH 7.5, 0.15 M NaCl. Anti-rabbit IgG-conjugated alkaline phosphatase diluted 1:10,000 wasused as a secondary antibody and 5-bromo-4-chloro-3-indolyl/nitro blue tetrazolium (Sigma Chemical) wasused for development.

M. Ehn et al. / Analytical Biochemistry 329 (2004) 11–20 15

Protein characterization: analysis of fractions pooledfrom puriWcation steps

Fractions from each puriWcation step showing highactivity in the luciferase assay and high purity on theSDS–PAGE gels were pooled for further experiments.The pooled samples were analyzed for total protein con-centration by the Bradford [27] method and for proteinpurity by SDS–PAGE.

Protein concentration. Total protein concentration infractions pooled from cation and anion exchange chro-matography was determined by Bradford [27] analysisusing BSA as a standard.

Protein purity. Samples from diVerent protein frac-tions were analyzed by SDS–PAGE. Gels used (Novex,San Diego, CA) were of the Tris–glycine type with poly-acrylamide gradients of 4–20% (Klenow-luciferase) and10–20% (SSB-luciferase). The amount of diVerent sam-ples loaded was adjusted so that each lane contained thesame amount of target protein. The gels were run underreducing conditions and stained with Coomassie brilliantblue R-350 according to the supplier's recommendations.Visual inspections and evaluation using QuantityOnesoftware (Bio-Rad, Hercules, CA) of the gels determinedthe purity of target protein in diVerent fractions.

SpeciWc luciferase activity. Luciferase activity wasmeasured, as described above, for pooled material fromcation and anion exchange chromatography. Afterrecording of the luciferase activity within a sample,400 ng of commercial luciferase was added to the sampleand the increase of the signal was compared to the sam-ple speciWc activity. By this operation, the molar amountof active luciferase within the sample was determined.This molar value was divided by the amount of thefusion protein in the sample that had been estimated byBradford and SDS–PAGE analysis.

Solid phase DNA analysis experiments

DNA immobilization and primer annealing. Threepicomoles of the biotinylated 90-mer OLIGOII or OLI-GOIII [18] was dissolved in 50 �l 10 mM Tris–HCl (pH8.3), 2 mM MgCl2, 50 mM KCl, 0.1% (v/v) Tween 20, andimmobilized onto streptavidin-coated superparamag-netic beads (Dynabeads M280; Dynal, Oslo, Norway).Immobilization was performed at room temperature for20 min and, after removal of unbound DNA, the beadswere resuspended in 10 �l annealing buVer (10 mM Tris–acetate, pH 7.75, 2 mM Mg–acetate) with 20 pmol of thesequencing primer, Malo3b [18]. Primer annealing wasperformed at 96 °C for 30 s followed by cooling to roomtemperature. Beads treated as real samples but with solu-tions devoid of oligonucleotide or primer were used asnegative controls.

Binding of fusion proteins to the immobilized DNA.The proteins were diluted in 40 �l of a solution

containing 0.1 M Tris–acetate, pH 7.75, 0.5 mM EDTA(dilution buVer). First, the protein molar amounts wereat least as high as that of the DNA template (3 pmol,supposing a 100% eVective DNA immobilization). Twoexperiments with diVerent relative molar amounts of theproteins aiming to investigate the inXuence on obtainedlight signal intensity were performed. Due to variationsin salt and protein concentrations, conductivity diVeredslightly between samples during protein–DNA bindingbut always remained between 5 and 14 mS/cm. The pro-teins were able to bind the DNA template by incubationat room temperature for at least 30 min. All proteinswere also incubated with negative control beads devoidof DNA for the same amount of time. Furthermore, onecontrol experiment in which 0.1 nmol of soluble lucifer-ase was incubated with DNA-coated beads was per-formed. Prior to DNA analysis, the supernatantcontaining unbound protein was removed and the beadswere resuspended in 13 �l of dilution buVer. The pre-pared samples were analyzed by a complete primerextension assay. The two proteins that gave the highestlight signal in these experiments (Zbasic-SSB-luciferaseand Klenow-luciferase) and Zbasic-Klenow-luciferasewere further studied by the same assay. In the followingexperiments, 30 pmol of fusion protein was used duringprotein binding. Moreover, experiments where the beadswere washed with 80�l dilution buVer after 30 min ofprotein binding were performed. The protein giving riseto the highest signal, Zbasic-SSB-luciferase, was used inpyrosequencing analysis of OLIGOII after immobiliza-tion according to the optimized procedure describedabove.

Complete primer extension assay. The samples wereadded to a 40-�l reaction mixture containing 20 mUrecombinant ATP sulfurylase, 0.1 M Tris–acetate, pH7.75, 0.5 mM EDTA, 5 mM Mg–acetate, 0.1% BSA, 1 mMDTT, 10�M adenosine 50-phosphosulfate (APS), 0.4 mg/ml polyvinylpyrrolidone, 100�g/ml D-luciferin, 2,5�MdGTP and dCTP, and 10 �M dTTP and �-thio-dATP(Amersham Biosciences). In the cases of SSB-luciferaseand Zbasic- SSB-luciferase, 10 U of the exonuclease-deW-cient (exo¡) Klenow DNA polymerase (Amersham Bio-sciences) was added to the reactions. In all experiments,1.4�g of puriWed luciferase (BioThema, Dalarö, Sweden)was added after a couple of minutes to certify thatabsence of light was due solely to absence of luciferase.To detect unspeciWc protein binding on DNA-free beads,1 pmol PPi was added. DNA polymerization was detectedin an automated single-tube pyrosequencer (Pyrose-quencing AB, Uppsala, Sweden).

Pyrosequencing using DNA-immobilized luciferase.The washed samples were added to a 40-�l reactionmixture containing 20 mU recombinant ATP sulfury-lase, 0.1 M Tris–acetate, pH 7.75, 0.5 mM EDTA, 5 mMMg–acetate, 0.1% BSA, 1 mM DTT, 10 �M APS,0.4 mg/ml polyvinylpyrrolidone, and 10 U of the

16 M. Ehn et al. / Analytical Biochemistry 329 (2004) 11–20

exonuclease-deWcient (exo¡) Klenow DNA polymerase(Amersham Biosciences), and 10 mU apyrase was addedto the reactions. Nucleotides were added iterativelyevery 8th min and DNA polymerization was detected inan automated single-tube pyrosequencer (Pyrosequenc-ing AB).

Results

To enable localization of light produced by luciferaseto DNA templates, two vectors encoding protein fusionsbetween the luciferase and a DNA-binding moiety (SSBor Klenow) were constructed. Both plasmids encodedfusion proteins with Zbasic as N-terminal puriWcationhandle, a 3C-protease cleavage sequence enabling post-puriWcation removal of Zbasic, and a DNA-bindingdomain (SSB or Klenow) followed by thermostableluciferase in the C terminus. Recombinant expression inE. coli followed by two steps of ion exchange chromato-graphic puriWcation with intervening removal of Zbasicresulted in highly pure protein preparations. Functionalanalysis of the proteins showed that they possess DNAaYnity and that the activity of the enzyme moieties isintact. Zbasic-SSB-luciferase gave rise to the highest sig-nal in those experiments. The enzyme was thereforeimmobilized on a DNA template and used as a light-producing enzyme in analysis of this genetic material bythe pyrosequencing technology. Several bases of the tem-plate could be sequenced with good data quality by useof the fusion protein. These results show the suitabilityof this easily prepared protein as enzymatic reagent forsequence determination of site-speciWcally attachedDNA templates.

Protein production, puriWcation, and characterization

The proteins were expressed in E. coli. After bacterialcultivation, the cells were disrupted and the soluble frac-tions (lane 1, Figs. 1C and 2C for Zbasic-SSB-luciferaseand Zbasic-Klenow-luciferase, respectively) were puriWedusing CIE. High yields of product binding to the matrixfor both proteins, 94 and 80% for Zbasic-SSB-luciferaseand Zbasic-Klenow-luciferase, respectively, were obtaineZbasic-SSB-luciferase was eluted by a stepwise increase ofsalt concentration (Fig. 1A). SDS–PAGE analysis showsthat during the 500 to 750 mM elution step, approxi-mately 19 mg of soluble Zbasic-SSB-luciferase at highpurity (80%) was obtained (lane 2, Fig. 1C). Approxi-mately 22 mg of soluble Zbasic-Klenow-luciferase waseluted from the column by use of a linear salt gradient(Fig. 2A). The coelution of 70-kDa degradation prod-ucts (identiWed by Western blot analysis; data notshown) resulted in a protein purity of 40% in this frac-tion (lane 2, Fig. 2C). To remove the Zbasic tag, site-spe-ciWc 3C-protease cleavage was performed at room

temperature overnight. The molar ratio between Zbasic-3C and target protein was 1:5 for Zbasic-SSB-luciferaseand 1:1 for Zbasic-Klenow-luciferase. Under these condi-tions, incomplete cleavage, and traces of degradationproducts was obtained for SSB-luciferase (lane 3,Fig. 1C). Attempts to increase the cleavage yield byincreasing the amount of 3C or cleavage time resulted inincreased degradation of SSB-luciferase (data notshown). On the contrary, 3C treatment of Zbasic-Klenow-luciferase resulted in almost complete cleavage of thetarget protein (lane 3, Fig. 2C). The cleavage mixtureswere puriWed by AIE and eluted by Xat salt gradients(Figs. 1B (SSB-luciferase) and 2B (Klenow-luciferase)).These puriWcations resulted in very pure protein prepa-rations of SSB-luciferase (98%) (lane 4, Fig. 1C) andKlenow-luciferase (93%) (lane 4, Fig. 2C).

Estimation of speciWc luciferase activity. The speciWcluciferase activity for each fusion protein was estimatedby a luminometric assay using internal standards ofcommercial luciferase as described under Materials andmethods. In the cases of Zbasic-SSB-luciferase, Zbasic-Klenow-luciferase, and SSB-luciferase, approximatelyhalf of the fusion proteins contained active luciferasedomains. Only Klenow-luciferase showed signiWcantlyhigher speciWc activity (90%). It should be stressed that,although this estimation is very rough, an indication offactors inXuencing luciferase signals in the DNA analy-sis experiments is obtained.

Solid phase DNA analysis experiments

Complete primer extension assay. To verify the activ-ity of the diVerent subunits within the fusion proteins,DNA extensions using ssDNA template attached toparamagnetic beads were performed. The beads withimmobilized primed DNA were incubated with thefusion proteins, Zbasic-SSB-luciferase, SSB-luciferase,Zbasic-Klenow-luciferase, and Klenow-luciferase, respec-tively. Beads lacking DNA were separately incubatedwith the fusion proteins and used as negative controls.Furthermore, one control experiment in which 0.1 nmolof soluble luciferase was incubated with DNA-coatedbeads was performed. The proteins were allowed to bindthe immobilized DNA and, after 30 min, unboundprotein was removed. A pyrosequencing mixture devoidof luciferase and, in the case of Klenow fusions, lackingKlenow was used. DNA synthesis would take place onthe primed template in the presence of polymerase sincethe mixture contained all four nucleotides. To providefull extension of the primed template, the enzyme apy-rase was also avoided. The reaction results in light emis-sion in the case of luciferase activity. The light intensitieswere plotted over time and the curves are shown in Fig. 3.In the cases of Klenow-luciferase and Zbasic-Klenow-luciferase, soluble Klenow was added to the reactionmixture at the time point where the light signal laid

M. Ehn et al. / Analytical Biochemistry 329 (2004) 11–20 17

steady at a plateau value. A small increase in signal wasachieved by this polymerase addition in the case of Kle-now-luciferase but not for Zbasic-Klenow-luciferase (datanot shown). For all fusion proteins, the light signal wasconsiderably increased by the addition of soluble lucifer-ase (data not shown). As clearly can be seen in Fig. 3, allfour fusion proteins have retained DNA-binding proper-ties and luciferase activity. Also, the Klenow fusions pos-sess polymerase activity. In the comparisons, Zbasic-SSB-luciferase gave the highest light signal and Klenow-lucif-erase gave the second highest. The light signal obtainedwhen DNA-coated beads were incubated with solubleluciferase was negible compared to those from experi-ments with the fusion proteins (data not shown). NounspeciWc protein binding to the DNA-free beads wasobserved except for one experiment with Zbasic-SSB-luciferase (data not shown). However, by increasing theconductivity in the protein solution during binding tothe DNA template, this unspeciWc interaction was inhib-ited. Nevertheless, an increased stringency for the pro-tein–DNA interactions was desirable and therefore theeVects of washing after protein binding were evaluated(Table 1). In these experiments, Zbasic-SSB-luciferase andKlenow-luciferase were used since they had given the

Table 1Comparison of light signals with and without washing of the beads

The plateau signals of the light intensity using the diVerent proteinsfor complete primer extension are shown. Measured light signal isgiven in arbitrary units. OLIGOIII is used in these experiments.

Protein used Light signal without washing

Light signal after washing

Zbasic-SSB-luciferase 300 240Klenow-luciferase 250 83Zbasic-Klenow-luciferase 55 38

highest light signals. Moreover, Zbasic-Klenow-luciferasewas included, enabling further investigation of the eVectof Zbasic on the Klenow domain in question of DNAaYnity and polymerase activity. Results from theseexperiments showed that washing after binding of theprotein to the template lowered the light signal for alltested proteins. However, the relative light reduction waslowest for Zbasic-SSB-luciferase and highest for Klenow-luciferase (Table 1). Although the detectable biolumines-cence for Zbasic-Klenow-luciferase was lower than that ofKlenow-luciferase, the wash reduced the light signalmore signiWcantly for the fusion protein devoid of Zbasic.Moreover, the light monitored in the complete primerextension was limited not by the polymerase activity forZbasic-Klenow-luciferase but by the luciferase concentra-tion. On the contrary, when soluble Klenow was addedto the Klenow-luciferase extension, the light signal wasincreased (Fig. 3).

Pyrosequencing using DNA-immobilized luciferase.Since Zbasic-SSB-luciferase gave the highest luciferasesignal in the extension assay after wash, this protein wasused as enzymatic reagent in pyrosequencing analysis ofthe used DNA template. In the experiment, the DNAwas incubated with 10 times molar excess of the proteinfor 30 min. After removal of the protein solution, thebeads were washed in dilution buVer and subjected topyrosequencing analysis where the nucleotides wereadded iteratively. For SNP analysis by pyrosequencing,four bases are suYcient for correct genotyping. Here, sixbases are sequenced, C-T-C-G-G-A, all correctly allo-cated (Fig. 4). To avoid signiWcant suppression of theluciferase signal, the amount of apyrase was 5 timeslower than customary for pyrosequencing [6,28]. Toguarantee suYcient nucleotide and ATP degradationbetween the base additions the cycle time was increased

Fig. 3. Complete primer extension assay performed on DNA immobilized on magnetic beads by use of the fusion proteins. The y axis denotes mea-sured light signal in arbitrary units and x axis time in seconds. (¡) DNA curves parallel to the x axis are reactions where all four fusion proteins havebeen incubated with DNA-free beads. The vertical arrow marks time point for addition of 5 units soluble Klenow. OLIGOII is used for the exten-sion.

18 M. Ehn et al. / Analytical Biochemistry 329 (2004) 11–20

from 1 to 8 min. However, despite the long cycle time,background peaks due to remaining nucleotides fromthe earlier steps can be seen in the pyrogram in Fig. 4.The width between peaks diVers slightly and the peakarea was therefore estimated (Table 2). This estimationshows that the integrated light signals were very similarfor all single-base incorporations and almost doubledfor the 2G extension. These results clearly illustrate thesuitability of Zbasic-SSB-luciferase as enzymatic reagentfor SNP or mutation detection on immobilized DNAtemplates.

Discussion

A strategy for production of two thermostable lucif-erase fusion proteins containing a DNA-binding domain(SSB or Klenow) and a puriWcation handle (Zbasic) thatcould enzymatically be removed was designed. Two pro-teins based on diVerent approaches for protein immobi-lization onto DNA were evaluated. The Wrst protein wasthe DNA polymerase generally used in the pyrosequenc-ing reaction, an exonuclease-deWcient (exo¡) variant ofthe Klenow fragment of DNA polymerase I [29]. Highlevels of this enzyme genetically fused to Zbasic hadearlier been obtained by production in E. coli followed

Table 2Comparison of integrated peak area from light signals obtained inanalysis of immobilized genetic material incubated with Zbasic-SSB-luciferase by use of the pyrosequencing method

The corresponding graph of light versus time is shown in Fig. 4.

Corresponding base Peak area (%)

C 100T 105C 1022G 180A 122

by CIE [14,30]. The second DNA-binding proteinexploited was SSB, which also had been eYcientlyexpressed in E. coli and selectively puriWed in a two-steppuriWcation protocol without aYnity tag [24]. The lucif-erase used was a thermostable variant of the enzymefrom P. pyralis containing four amino acid substitutions.Also, our primer design introduced a Wfth amino acidshift in the C-terminal L550 ! V. Since the C terminus ofluciferase has not been reported to interfere with theactivity or the structure of the protein and the twoamino acids are chemically similar, the penta-mutantluciferase was used in the experiments. The two proteinswere eYciently expressed in 500-ml shake Xask cultiva-tions using E. coli, giving approximately 200 nmol forboth proteins corresponding to 19 and 28 mg of solubleZbasic-SSB-luciferase and Zbasic-Klenow-luciferase, respec-tively. The proteins were selectively and eYciently puri-Wed by CIE under mild conditions aiming to spare theactivity of the enzymatic domains. The Zbasic tag wasremoved by enzymatic cleavage using the 3C-protease[12,13]. Subsequent puriWcation of the cleavage productsby AIE resulted in highly pure SSB-luciferase and Kle-now-luciferase, although the yields after cleavage andAIE were moderate. Although further optimization ofthese two steps probably could increase the proteinyields, extensive work was not put into optimization ofthe downstream protein processing eYciency but ratherwas put into obtaining proteins of high purity. All fusionproteins showed similar speciWc luciferase activity afterpuriWcation except for Klenow-luciferase, whose valueswere signiWcantly higher than those of the other variants.Hence, larger diVerences in functional analysis based onbioluminescence of the other fusion proteins are proba-bly due to variations in template binding. The enzymaticactivities and DNA template aYnity for the fusion pro-teins were investigated by an assay based on biolumino-metric detection of complete extension of primed

Fig. 4. Pyrosequencing analysis of DNA immobilized on magnetic beads using Zbasic-SSB-luciferase as light harvesting reagent. The y axis denotesmeasured light signal in arbitrary units and the letters under the x axis give the nucleotide addition order. Small background peaks (T and C) can beseen in the pyrogram due to the low concentration of apyrase.

M. Ehn et al. / Analytical Biochemistry 329 (2004) 11–20 19

template. Two separate experiments with diVerentamounts of the proteins and slightly diVerent conductiv-ity in the samples were performed. Although the relativemolar amounts of the four proteins diVer vastly betweenthe two experiments, the amount of protein bound to theDNA should be the same due to incubation with satu-rating amounts of protein. This is conWrmed by the factthat the relative order of the diVerent proteins withregard to light intensities was almost identical in thediVerent experiments. Hence, the diVerent signal heightsobtained from diVerent proteins should be due to varia-tions in aYnities of the proteins for ssDNA. As clearlycan be seen in Fig. 3, Zbasic-SSB-luciferase binds mosteYciently to the template and therefore results in higherlight signal intensity. The second highest light intensitywas obtained from Klenow-luciferase and, belowthis, SSB-luciferase and Zbasic-Klenow-luciferase showedapproximately the same signal intensity. In one of theexperiments unspeciWc interactions between the Zbasic-SSB-luciferase and the DNA-free beads were detected.However, by increasing the conductivity in the sampleduring protein immobilization, this protein stickingeVect was eYciently prevented. The additions of com-mercial luciferase and DNA polymerase after extensionby the fusion proteins indicate that the luciferase activityof all fusion proteins is the major limiting factor for lightsignal intensity. Moreover, an increase in bioluminomet-ric signal was obtained by polymerase addition to Kle-now-luciferase but not to Zbasic-Klenow-luciferase. Thesedata indicate that Zbasic might increase the interactionbetween the DNA template and the protein and therebycould facilitate the polymerase activity. Fig. 3 also indi-cates that the positive charges on the Zbasic domainmight increase the aYnity of SSB-luciferase for ssDNA.Therefore, the only essential downstream processing ofthe proteins before use could be the CIE, which is veryselective, eYcient, mild, cost-eVective, and easy to scaleup. These results suggest that high amounts of DNA-binding Luciferase can be obtained by expression in E.coli followed by rapid and mild puriWcation. Washing ofthe beads after binding of the proteins to the immobilizedDNA shows that Zbasic-SSB-luciferase has the strongestinteraction to the DNA (Table 1). Therefore, this con-struct was used for sequencing of immobilized DNA. Thepyrogram in Fig. 4 shows that Zbasic-SSB-luciferase canbe used for analysis of immobilized DNA templates bythe pyrosequencing technology. To date, the alreadydeveloped pyrosequencing method using soluble enzymesis working better due to higher signals. However, theavailability of more sophisticated detection systems, suchas highly sensitive CCD cameras, could most likelyenable the use of the luciferase fusion proteins in nano-liter micromachined Wlter chambers. In this approach, thereaction chamber contains streptavidin-coated beadswith immobilized DNA template to which the luciferaseis bound. The other reaction components are Xown

through the chamber so that no dilution is obtained byreagent addition. This approach may improve thethroughput and decrease the cost of SNP analyses in thefuture. Also, further development of the system for SNPanalysis on DNA arrays can be envisioned.

Acknowledgment

The authors thank Dr. L. Tisi for kindly providing theplasmid pPWLTx4.

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