rapid mutagenesis and purification of phage rna …...protein expression and purification 9,...

PROTEIN EXPRESSION AND PURIFICATION 9, 142–151 (1997)ARTICLE NO. PT960663

Rapid Mutagenesis and Purificationof Phage RNA Polymerases

Biao He, Mingqing Rong, Dmitry Lyakhov, Heidi Gartenstein, George Diaz,Ray Castagna, William T. McAllister, and Russell K. DurbinDepartment of Microbiology and Immunology, Morse Institute for Molecular Genetics, State University of New York,Health Science Center at Brooklyn, 450 Clarkson Avenue, Box 44, Brooklyn, New York 11203-2098

Received May 28, 1996, and in revised form August 22, 1996

based expression systems that allow rapid mutagenesisWe have developed plasmid-based expression sys- and purification of T7 and other phage RNA polymer-

tems that encode modified forms of T7 RNA polymerases.ase (RNAP) having 6–12 histidine residues fused to the Previous methods for purification of T7 RNAP haveamino terminus. The histidine-tagged RNAPs (His-T7 included selective precipitation with salt, size-exclu-RNAPS) are indistinguishable from the wild-type (WT) sion chromatography, chromatography on ion exchangeenzyme in nearly all biochemical assays. Similar plas- columns, and affinity chromatography on columns suchmids that encode His-tagged T3 and SP6 RNAPs have as Affi-Gel blue or GTP–agarose (1–9). In general,also been constructed. To facilitate site-directed muta- these methods are time consuming and require dedi-genesis of the RNAP gene, the size of the target plas- cated columns and chromatography equipment for eachmid was minimized by using T7 RNAP itself as a se- enzyme preparation. We were interested in rapid meth-lectable marker. BL21 (DCAT4) cells (which carry a ods for simultaneous purification of multiple sampleschromosomal copy of the chloramphenicol acetyl- of RNAP, without the need for dialysis or de-saltingtransferase cat gene under control of a T7 promoter) between chromatography steps. We have developedare resistant to chloramphenicol when functional T7

two such methods, one for purification of conventionalRNAP is expressed, thus allowing the selection and(unmodified) RNAPs, the other for RNAPs that havemaintenance of the target plasmid in these cells. Muta-been modified to include a histidine tag at their aminogenesis is accomplished by denaturing the plasmid,termini. Both methods are rapid and give enzymes ofannealing mutagenic DNA primers, and repairing thehigh quality which are suitable for use in transcriptionplasmid with T4 DNA polymerase. Two DNA primersassays and biochemical characterizations.are used: one corrects a defect in the bla gene, the

A number of methods have been devised for engi-other introduces the desired mutation into the RNAPneering proteins with affinity tags and the subsequentgene; 30–85% of the ampicillin-resistant transformantspurification of the modified proteins by chromatogra-carry the desired mutation in the RNAP gene. By using

BL21 (DCAT4) cells as a recipient for transformation phy over a ligand-bearing column. In the His-tagthe functional integrity of the RNAP gene may conve- method the gene that encodes the desired protein isniently be monitored by assessing the level of chloram- modified to encode six or more adjacent histidine resi-phenicol resistance in vivo. Methods for rapid, simulta- dues which, if surface-exposed, result in a protein withneous purification of multiple samples of modified a high affinity for Ni2/ ions that are immobilized on a(His-tagged) and conventional RNAPs are described. column (10,11). The desired protein may then be elutedTogether, these developments greatly enhance our with an appropriate concentration of imidazole.ability to characterize this important class of enzymes. To adapt this approach to the purification of T7 RNAq 1997 Academic Press polymerase we took advantage of the observation that

T7 RNAP can tolerate substitutions at the amino ter-minus. Previous modifications that do not disruptRNAP activity include the attachment of a nuclear lo-calization signal from SV40 (12) and naturally oc-In our work, it is necessary to generate and charac-

terize a large number of mutant T7 RNA polymerases. curring fusions that occur in T7 deletion mutants suchas C5a (13). In the crystal structure of T7 RNAP theTo facilitate these studies we have developed plasmid-

142 1046-5928/97 $25.00Copyright q 1997 by Academic Press

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MUTAGENESIS AND PURIFICATION OF PHAGE RNA POLYMERASES 143

amino terminus is observed to project out from the a T7 promoter, the activity of the modified RNAP invivo may readily be determined by assessing the level‘‘back’’ surface of the enzyme, away from the DNA bind-of chloramphenicol resistance of the transformantsing cleft and the active site (14), which may explain(12,13).why the RNAP tolerates modifications in this region.

These developments allow us to rapidly purify T3,We have found that the T3 and SP6 RNAPs are alsoT7, and SP6 RNAPs, and to mutagenize the respectiveable to tolerate the fusion of a histidine tag at theirRNAP genes with great facility.amino termini.

To facilitate mutagenesis of the RNAP gene we uti-MATERIALS AND METHODSlized a plasmid-based system that involves two DNAPlasmids and Synthetic DNAprimers. One of the primers incorporates the desired

mutation into the RNAP gene, and the second primer Plasmid manipulations were carried out using stan-corrects a defect in the b-lactamase (bla) gene that is dard methods (15); sequences of all plasmids are avail-also present in the plasmid. Selection for bla/ cells able upon request. Synthetic DNA and RNA oligomersamong the transformants results in a high frequency (A–F) were prepared by Macromolecular Resourcesof the desired mutation in the RNAP gene. Through (Boulder, CO) or in the SUNY-Health Science Centerthe use of a bacterial strain that carries the chloram- Facility and purified by HPLC or Sep-Pak (Waters) col-

umn chromatography.phenicol acetyltransferase gene (cat) under control of

Oligo A 5*-CTATGTATTCTGTAACTAGATTGC-3 * (LG3)

Oligo B 3 *-GATACATAAGACATTGATCTAACG-5* (LG4)

C AOligo C A GCGCTTTAATTATGCTGAGTGATATCCCTCT-5* (MR18)

A CGCGAAATTAATACGACTCACTATAGGGAGA-3 *C A

C AOligo D A CGCGAAATTAATACGACTCA-3 * (MR19)

A GCGCTTTAATTATGCTGAGTGATATCCCTCT-5*C A

Oligo E 5*-GGGAAGUCUGUACCAGACGU-3 * (MR15)

Oligo F 5*-GGGAAGTCTGTACCAGACGT-3 * (MR16)

Propagation and Growth of Bacterial Cultures terial culture is harvested by centrifugation; the pelletmay be frozen until further use.

A culture of BL21 (7) carrying the desired plasmidis streaked on LB agar containing 60 mg/ml ampicillin Rapid Purification of His-Tagged T7 RNA Polymerase[LB / Amp (15)] and, if pDM1.1 is present, 10mg/ml

1. Buffers and reagents. Charge buffer is 50 mMkanamycin. A fresh colony is used to inoculate 2 ml ofNiSO4. Binding buffer is 150 mM K Glutamate, 30 mMLB / Amp broth; the liquid culture is then incubatedHepes, 0.05% Tween 20, and 5 mM imidazole. Washovernight (16 h) without shaking at 307C. On the fol-buffer is binding buffer containing 20 mM imidazole.lowing day, 30 ml of fresh LB/ Amp in a 125-ml Erlen-Elution buffer is binding buffer containing 100 mM im-myer flask is inoculated with 0.5 ml of the overnightidazole. PMSF (0.2 M phenylmethysulfonyl fluoride,culture and incubation is continued with vigorous aera-Sigma P-7626) is prepared in ethanol and stored attion at 377C until the culture achieves an optical den-0207C. Lysozyme (10 mg/ml in distilled water) is storedsity of OD600 Å 0.4–0.6 (usually 3–4 h). At that time,at 0207C in individual use aliquots.isopropyl-b-D-thiogalactopyranoside (IPTG) is added to

a final concentration of 0.4 mM, and incubation is con- 2. Cell lysis. The cell pellet is resuspended in 0.8tinued for 4 h. The conditions of culture propagation ml binding buffer and 1/10 vol of lysozyme and 1/200are important to ensure plasmid maintenance and high vol of 0.2 M PMSF are added. The sample is held atlevels of induction; typically, up to 30% of total protein room temperature for 15 min, freeze-thawed one time,in the crude lysate is phage RNAP, as judged by gel and sonicated for two 30-s intervals on ice. (We use

a Kontes microprobe tuned for maximal power.) Theelectrophoresis. Following induction, 20 ml of the bac-


HE ET AL.144

sample is clarified by centrifugation at 15,000 rpm for Rapid Purification of Unmodified T7 RNAPolymerase15 min.

3. Column chromatography. His-Bind resin (Nova- 1. Buffers and reagents. Harvest buffer is 50 mMgen) is resuspended by swirling, and 1.0 ml of the slurry Tris-HCl, pH 8.0, 2 mM EDTA, 20 mM NaCl, 1 mMis added to a disposable Bio-Rad polyprep column (Cat. dithiothreitol (DTT), and 0.05% Tween 20. Buffer A isNo. 731-1550) fitted with a 23-gauge needle. After set- 40 mM KHPO4, pH 7.7, 1 mM EDTA, 1 mM DTT, 5%tling, this provides a column volume of ca. 0.7 ml, and glycerol, and NaCl as indicated. Whatman DE52 cellu-a flow rate of ca. 1 ml/6 min. lose and P11 phosphocellulose are precycled in alter-

The column is washed successively with 6 ml of wa- nate washes of HCl and NaOH and adjusted to pHter, 10 ml of charge buffer, and 6 ml of binding buffer. 7.7 as recommended by the manufacturer. Fines areThe sample is loaded and washed in with 4 ml of bind- removed by allowing the slurry to settle for 3 min, anding buffer. The column is then washed with 10 ml of buffer A plus 25 mM NaCl (DE52) or 50 mM NaCl (P11)wash buffer and eluted with two 2-ml aliquots of elu- is added to adjust the slurry to 120% of settled bed

volume.tion buffer. The peak fraction(s) may be identified byspotting 10 ml of each fraction on 3MM Whatman filter 2. Sample preparation and chromatography. Cellpaper and staining with 0.025% Coomassie blue in 25% pellets are resuspended at 07C in 1 ml harvestingisopropanol, 10% acetic acid, and then destaining with buffer, lysozyme and PMSF are added, and the samples5% isopropanol, 10% acetic acid (8). Most of the RNAP are sonicated as described above. Deoxycholate isis usually found in the first fraction. At this point, the added to 0.1% and the samples are incubated on ice forenzyme is 95% pure, as judged by SDS–PAGE, essen- 20 min. Proteins are precipitated by the addition of 3.8tially free of most nucleases, and suitable for use in M ammonium sulfate (pH 7.8) to a final concentrationtranscription assays. Enzyme concentration is deter- of 0.2 M. The mixture is incubated on ice for 10 minmined by measuring the optical density at 280 nm us- and centrifuged at 10,000 rpm for 20 min, and the su-ing a molar extinction coefficient of e280 Å 1.4 1 105 M01 pernatant is collected. Nucleic acids are precipitatedcm01 (5). Typical yields from a 20-ml culture are 0.5 to by the addition of 0.1 vol of 5% polyethyleneimine1.5 mg of purified RNAP in a volume of 2.0 ml. (PEI), pH 7.9, followed by incubation on ice for 20 min

The sample may be stored at 0207C after the addi- and centrifugation at 10,000 rpm for 20 min. To removetion of glycerol to 50% (v/v) with no significant loss PEI (which otherwise interferes with subsequent chro-of activity for up to 6 months. Alternatively, imidaz- matography steps) the supernatant is mixed with 0.5ole may be removed and the enzyme placed in a more vol of a slurry of phosphocellulose (see above). Afterconventional buffer by absorption and elution from gentle mixing at 47C for 10 min, the sample is centri-a 0.5-ml phosphocellulose column, as described be- fuged at 10,000 rpm for 10 min and the conductivity oflow, or through the use of spin columns (Centricon, the supernatant is adjusted to that of buffer A plus

25 mM NaCl by dilution with buffer A. The sample isAmicon).

TABLE 1

Properties of His-T7 RNAP Expression Vectors

FlankingPlasmid Leader sequencea RNAP Promoterb sitesc lacIq d blae

pBH116 MGSS(H)6LVPRGSHMLEMNTIN. . . T7 Plac UV5 NcoI, XhoI 0 0pBH117 MGSS(H)6LVPRGSHMLEMNTIN. . . T7 Plac UV5 NcoI, XhoI 0 /pBH161 MGSS(H)6LVPRGSHMLEMNTIN. . . T7 Plac UV5 NcoI, XhoI / /pDL19 M(H)6MHTIN. . . T7 Plac NsiI / /pDL21 M(H)6M(H6)MHTIN. . . T7 Plac NsiI / /pBH118 MGSS(H)6SSGLVPRGSHMLEMNIIE. . . T3 Plac NcoI, XhoI / /pBH176 MGSS(H)6SSGLVPRGSHMLEMQDLH. . . SP6 Plac NcoI, XhoI / /pDL18 M(H)6MNIIE. . . T3 Plac NcoI, XhoI / /

a The N-terminal sequence of the fusion protein. The original initiation codon of the RNAP gene is double-underlined; a potential thrombincleavage site is single-underlined. Note that in pDL19 and pDL21 the second amino acid in the RNAP has been changed from the WTresidue (Asn) to His.

b The promoter driving expression of the RNAP gene is either Plac or PlacUV5, as indicated.c These unique sites may be used to excise the His leader from the plasmid.d Plasmids that do not carry the lacIq gene are best maintained in a host cell that also carries pDM1.1 as a source of lac repressor.e The bla gene in pBH116 is defective due to a 4-bp deletion.



clarified by centrifugation at 10,000 rpm for 3 min, andloaded onto a 2-ml DE52 cellulose column which hasbeen equilibrated with 7 vol of buffer A plus 25 mM

NaCl. The column is eluted with 3 vol of buffer A plus125 mM NaCl, collecting 1-ml fractions. Peak fractions(identified by gel electrophoresis) are loaded directlyonto a 0.5-ml phosphocellulose column that has beenequilibrated with 5 vol of buffer A plus 50 mM NaCl.The column is washed with 5 vol of buffer A plus 100mM NaCl and the RNAP is eluted by the addition of 3vol of buffer A plus 400 mM NaCl, collecting fractionsof 0.5 ml. Peak fractions are identified by spotting onWhatman 3MM filter paper as described above.

Transcription Assays

Unless otherwise indicated, transcription reactionsare carried out in a volume of 20 ml containing 30 mM

Hepes, pH 7.8, 100 mM potassium glutamate, 15 mM

magnesium acetate, 0.25 mM EDTA, 1 mM DTT, 0.05%Tween 20, 0.4 mM GTP, CTP, UTP, and ATP, [a-32P]-ATP (0.1 mCi), 1 mg of plasmid template (as indicated),and 20 ng of RNAP (16). The reactions are prewarmedat 377C for 2 min and initiated by the addition of RNAP.

For analysis of abortive initiation products, reactionscontain 20 mM Tris-HCl, pH 7.9, 8 mM MgCl2, 0.1 mM

EDTA, 1 mM DTT, 0.05% Tween 20, and 1 mg of pLM45(17) digested with SmaI. The runoff (86 nt) and abor-tive products are resolved by electrophoresis in 20%polyacrylamide gels (15). The ability of the enzyme to‘‘stutter’’ at a promoter that initiates with the sequence5* GGG . . . is monitored by production of a G-ladderin reactions that contained 0.4 mM [g-32P]GTP as thesole substrate (18).

Determination of Specific Activity

Reactions containing 20 ng T7 RNAP are incubatedunder standard conditions using pLM45 as template.At 5, 10, 20, and 40 min, duplicate 5-ml aliquots arespotted onto Whatman 3MM filter paper disks whichhave been presoaked in 10% trichloracetic acid (TCA)and dried. The filters are batch-washed in 10% TCAand TCA-precipitable material is determined by count-ing in a toluene-based scintillation fluid. Specific activ-ity is calculated as nanomoles UMP incorporated perhour at 377C per milligram of enzyme (1) as determinedfrom the linear portion of the response curve.

FIG. 1. Structure of His-tagged RNAP vectors. pBH116 is the tem- Determination of Nuclease Activityplate plasmid for mutagenesis of the T7 RNAP gene; the bla gene inthis plasmid is defective due to a 4-bp deletion. pBH161 carries a func- Enzyme preparations are incubated for 60 min withtional copy of the bla gene and the lacIq gene as a source of lac repressor; the polynucleotide substrate indicated in reactionthis plasmid is similar in its organization to pAR1219 (4,22). Plasmids buffer lacking rNTPs. The enzyme is present at eitherpBH118, pBH176, DL19, and DL21 carry His-tagged versions of T3,

the normal concentration (20 ng or 8 units per 20-mlSP6, or T7 RNAPs as indicated; the organization of these plasmids isreaction) or 10 times this concentration. Following in-similar to that of pCM56 (23). See Table 1 for a more detailed descrip-

tion of the His-tagged RNAPs in these plasmids. cubation, the integrity of the substrate is examined by


HE ET AL.146

FIG. 2. Purification of unmodified and His-tagged RNAPs. RNAP was purified as described under Materials and Methods; individualfractions were analyzed by electrophoresis in polyacrylamide gels followed by staining with Coomassie blue. (A) Conventional (unmodified)RNAP: lane 1, markers (Bio-Rad, lower molecular weight); lane 2, lysate; lane 3, DEAE-cellulose flowthrough and wash; lane 4, eluate fromDEAE-cellulose; lane 5, phosphocellulose flowthrough; lane 6, phosphocellulose wash; lane 7, phosphocellulose eluate. (B) His-tagged RNAP:lane 1, markers; lane 2, lysate; lane 3, His-bind flowthrough; lanes 4 and 5, His-bind wash; lane 6, His-bind eluate.

electrophoresis. In control experiments, we have deter- sequence from 017 to /6; oligomer D is a partiallymined that these assays can readily detectõ1004 units single-stranded promoter sequence (19). The oligomersof DNase (T4 DNA polymerase) and õ1 pg of RNase A were labeled with [g-32P]ATP by T4 polynucleotide ki-activity (data not shown). nase and purified by gel electrophoresis (15). Binding

To detect DNA endonuclease and nicking activity, assays were carried out as previously described (19,20).the substrate is either 1 mg of pUC19 or 1 mg of a HinfI Reactions (20 ml) contained 10 mM sodium phosphatedigest of pUC19 DNA; the products are resolved by buffer, pH 7.8, 1 mM EDTA, 20 mM NaCl, 4% glycerol,electrophoresis in 0.8% agarose gels or 8% denaturing 3000 cpm (approximately 1–2 ng) of the appropriatepolyacrylamide gels (15). oligomer, and RNAP as noted. In reactions involving

To detect RNase activity, a labeled RNA substrate is oligos C and D, 0.5 mg single-strand salmon sperm DNAprepared by transcription of a HindIII digest of pET15b was added as a nonspecific competitor. After incubation(Novagen) under standard conditions. The reaction at 307C for 10 min the samples were loaded onto a 8%products, which include a 433-nt runoff product and polyacrylamide gel in 0.51 TBE (45 mM Tris-borate, 1a 249-nt product arising from termination at TF, are mM EDTA) that had been preelectrophoresed for 1 hisolated by phenol-chloroform extraction and precipita- at 150 V. The samples were resolved by electrophoresistion with ethanol (15). The RNA (ca. 3000 cpm) is incu- at room temperature at 150 V for 2 h.bated with enzyme preparations as described above, The ability of the RNAP to bind to single-strandand the products are resolved by electrophoresis in 8% oligomers of RNA (oligo E) or DNA (oligo F) was deter-polyacrylamide gels (15). mined by means of a gel retardation assay, as described

To detect single-stranded exonuclease activity, oligo- above, but in the absence of competitor DNA.nucleotide A above is labeled with [g-32P]ATP using T4polynucleotide kinase, and purified by electrophoresisin a 20% polyacrylamide gel in 40 mM Tris-acetate, 1 MutagenesismM EDTA buffer (15). Approximately 3000 cpm (ca. 0.1ng) of labeled substrate are utilized per reaction. Site-directed mutagenesis was carried out following

To detect double-stranded exonuclease activity, 200 the USE (Unique Site Elimination) protocol (Phar-ng labeled oligo A is annealed with 300 ng oligo B and macia Biotech). The template for mutagenesis wasthe double-stranded form is purified and utilized as a pBH116, purified by isopycnic banding in a gradient ofsubstrate as described above. CsCl (15). The bla gene repair oligomer had the se-

quence 5*-CGTGACACCACGATGCCCGCGGCAATG-Binding of DNA and RNA Oligomers GCAACAACGTT. The repaired plasmid was trans-

formed into DH5a (15) and transformants were platedOligomers C and D form stable stem–loop struc-tures. Oligomer C contains a consensus T7 promoter in LB/Amp plates.



FIG. 3. Quality of His-tagged RNAP preparations. To determine potential nuclease contamination, 20 or 200 ng of RNAP was incubatedwith the substrate indicated at 377C for 1 h in transcription buffer. The integrity of the substrate was then analyzed by electrophoresis asindicated under Materials and Methods. RNAP preparations were as follows: HIS, His-T7 RNAP; CON, conventional (unmodified RNAP);COM, commercial; C, control (no RNAP). H2O and BUF indicate incubation of the substrate with distilled water or transcription buffer,respectively. Substrates were as follows: (A) Supercoiled plasmid (pUC19). (B) Double-strand DNA (a HinfI digestion of pUC19). (C) A 24-nt single-strand oligomer of DNA. (D) A 24-bp double-stranded DNA fragment (as above, but annealed to its complement). (E) RNA; twospecies are present: the smaller 249-nt product arises from termination at TF and has a stem–loop structure at the 3* end, and the largeris a 433-nt read-through product having an internal stem–loop structure.

RESULTS six contiguous histidine residues and also specifies athrombin cleavage site. Unique NcoI and XhoI sitesConstruction of Plasmid Vectors permit excision of the leader sequence from the vector,

A number of histidine-tagged derivatives of T7 RNAP allowing the original start codon of the RNAP gene tohave been constructed (Table 1 and Fig. 1). Each of be placed at an appropriate distance from the ribosomethese has advantages in certain circumstances. binding-site. Although expression of the His-T7 RNAP

Plasmids pBH116, pBH117, and pBH161 carry a gene is under control of the lac UV5 promoter, pBH116His-tagged T7 RNAP gene in which the leader sequence and pBH117 do not carry a lacIq gene, and hence there

is a significant level of background expression of T7is derived from pET15b (Novagen). This leader encodes


HE ET AL.148

FIG. 4. Biochemical properties of His-tagged RNAP. The abilities of His-tagged and conventional (unmodified) T7 RNAPs to carry outkey aspects of the transcription process were determined as described under Material and Methods. (A and B) Promoter binding. Syntheticoligomers containing a T7 fully double-stranded promoter sequence (oligo C, panel A) or a partially double-stranded promoter (oligo D, B)were incubated with no RNAP (lane 1) or with increasing amounts (100, 200, 400, or 600 ng) of His-T7 RNAP (lanes 2 to 5) or conventionalRNAP (lanes 6 to 9) and the reactions were resolved by electrophoresis in a nondenaturing gel. The positions of bound and free oligomersare indicated. (C) Binding of single-strand RNA; reactions were carried out as described for (A) except that the ligand was a 20-nt oligomer

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RNAP from these plasmids. To provide tight control of Quality of RNAP Preparationsexpression of the RNAP gene, a compatible plasmid

We performed several biochemical assays to deter-that carries the lacIq gene on a p15A replicon is utilizedmine the quality of the His-tagged enzyme and its abil-[pDM1.1 (21)].ity to carry out various steps in the transcription cycle.Plasmids pBH161, pBH118, and pBH176 carry His-In all of these assays the enzyme was compared withtagged versions of T7, T3, or SP6 RNAP under controlWT RNA polymerase (that is, RNAP without a histi-of the lacUV5 promoter, but unlike the series above,dine-tag) prepared by conventional methods in our lab-these vectors also carry the lacIq gene and may be in-oratory, and with a limited number of commercial prep-duced and propagated essentially as pAR1219, pCM56,arations. The specific activity of the enzyme wasand pSR3 (8,22,23). These are therefore the plasmids300,000–400,000 units/mg, which is close to the theo-of choice for routine production and purification of his-retical maximum for a preparation in which all enzymetidine-tagged versions of these RNAPs.molecules are active (18).pDL19 and pDL21 carry different versions of His-

To detect possible nuclease contamination, we incu-tagged T7 RNAP in which a leader sequence that lacksbated RNAP preparations with a variety of substratesthe thrombin cleavage site and specifies either 6 orunder standard conditions. These substrates included:12 histidine residues is utilized. These vectors haveclosed circular plasmid DNA (to look for endonucleaseadvantages in situations in which an additional throm-and nicking activity), restriction fragments of DNA (tobin cleavage site in the enzyme is not desired, or inlook for endonuclease and exonuclease activity), end-which a higher affinity for the Ni2/ affinity column islabeled double-stranded and single-stranded oligomersneeded. pDL18 carries a gene coding for a similar ver-of DNA (to look for exonuclease activity), and a 400-ntsion of T3 RNAP.RNA transcript having an internal stem–loop struc-ture (the T7 terminator, TF) to look for RNase activity.

Purification of Histidine-Tagged and Unmodified Reactions were carried out for 60 min using either theRNA Polymerases usual concentration of T7 RNAP (20 ng or 8 units per

20-ml reaction) or 10 times this amount (see Fig. 3).Detailed protocols for rapid, small-scale purificationof histidine-tagged and conventional enzymes are pre- No significant nuclease activity was detected in the

histidine-tagged preparations, and little nuclease ac-sented under Materials and Methods.In the protocol for purification of the His-Tagged tivity was observed in most other preparations except

under the most stringent conditions.RNAP the volume of the Ni2/ affinity column has beenkept to a minimum to conserve resin during the prepa- To determine whether His-T7 RNAP exhibited any

novel or unusual biochemical properties, we examinedration of multiple samples, and to allow rapid chroma-tography. For preparative-scale isolation investigators the ability of the polymerase to carry out individual

steps in the transcription cycle (Fig. 4). These included:should increase the size of the column in proportion tothe sample volume. RNAP prepared by this method is specific activity, ability to bind to promoter-containing

DNA fragments or nonspecifically to single-strandedestimated to be greater than 95% pure as judged bygel electrophoresis (Fig. 2). Typical yields are 0.5–1.5 RNA, ability to synthesize long products or short (abor-

tive) initiation products, and ability to recognize termi-mg from 20 ml of culture. The procedure outlined israpid and convenient. We typically purify 8–10 sam- nation signals. Within experimental error, His-T7

RNAP performed in a similar fashion to conventionallyples of RNAP at a time, and the total purification, in-cluding lysis of the cells, requires less than 5 h. purified, unmodified T7 RNA polymerase in all of these

assays. A uv spectrum analysis of both enzyme prepa-The protocol for purification of conventional RNAPis also rapid, and may be completed in 1 day. However, rations indicates that a significant amount of cellular

DNA is present in the His-T7 RNAP sample. This doesas more steps are involved it is less convenient thanthe histidine-tagged protocol. The yield and quality of not appear to affect the properties of the enzyme prepa-

ration, but if desired this DNA may easily be removedthe enzyme are comparable to that described above.

of single-strand RNA (oligo E). (D) Synthesis of short RNAs and abortive initiation products. Plasmid pLM45, which contains a consensuspromoter, was digested with SmaI to result in the production of a short (83 nt) run-off product in the presence of all four ribonucleosidetriphosphates as substrate (lanes 1 and 2) or in the production of a ‘‘G-ladder’’ in the presence of GTP as substrate (lanes 3 and 4) (25).Products of transcription by His-T7 RNAP and conventional RNAP were resolved by electrophoresis in a 20% polyacrylamide gels. Thepositions of run-off and abortive products are indicated. (E) Synthesis of long RNAs and termination. Plasmid pLM44, which contains theT7 termination signal, TF, or pET15b, which contains the PTH terminator, were digested with XbaI and HindIII, respectively, to result inthe production of termination (T) and run-off (R) products, as indicated. The products synthesized by conventional (WT) and His-taggedRNAP (HIS) were resolved by electrophoresis in 6% polyacrylamide gels. The efficiency of termination (fraction of total RNA as terminatedRNA) is indicated below each lane.


HE ET AL.150

by passage of the sample over a small column of phos- be of excellent quality as assessed by their high specificactivity and lack of nuclease contamination.phocellulose, as described for purification of the con-

ventional enzyme. In addition, we have devised plasmid templates thatallow rapid mutagenesis of the T7 RNAP gene and thescreening of biological activity of the modified enzymeRapid Mutagenesis of the T7 RNAP Genein one convenient step. These developments greatly en-We also wished to develop an efficient method to gen- hance our ability to carry out structure and functionerate mutants of T7 RNAP that could be purified by studies on this enzyme. Similar vectors that encodethe His-Tag technology. The method of mutagenesis is His-tagged T3 and SP6 RNAP and a host cell thatbased upon a two-primer approach in which one primer allows a determination of T3 RNAP activity (24) haveintroduces the desired mutation into the RNAP ‘‘tar- been constructed.get’’ gene and the other mutation confers a selective The availability of His-tagged forms of the phagephenotype on the plasmid. It is therefore similar to the RNAPs will likely prove useful for a variety of applica-pSELECT (Promega) and USE (Pharmacia) protocols. tions, such as solid state nucleic acid synthesis (usingIn such plasmid-based mutagenesis systems, the size immobilized enzyme), controlled substrate addition ex-of the plasmid is important to the efficiency of muta- periments (e.g., template ‘‘walking’’), and purificationgenesis, and it is desirable to keep the plasmid as small of amino terminal fragments of the RNAP.as possible. To accomplish this, we utilized T7 RNAP

itself as a selectable marker. In pBH116, the T7 RNAPACKNOWLEDGMENTSgene is under control of the lacUV5 promoter (PlacUV5).

In BL21(DCAT4) cells [in which expression of a chro-These studies were supported by NIH Grant GM38147. This work

mosomal copy of the chloramphenicol acetyltransferase has been submitted to the State University of New York in partial(cat) gene is under control of a T7 promoter] the level fulfillment of the requirement for the doctoral degree of Biao He.of T7 RNAP expression provided by pBH116 rendersthe cell resistant to chloramphenicol. pBH116 also car-

REFERENCESries an ampicillin-resistance gene (bla) that is defectivedue to a 4-bp deletion. The second primer corrects this 1. Chamberlin, M., McGrath, J., and Waskell, L. (1970) New RNA

polymerase from Escherichia coli infected with bacteriophagedefect, while the first primer introduces the desiredT7. Nature 228, 227–231.mutation in T7 RNAP. Mutagenesis is accomplished

2. Chamberlin, M. J., Kingston, R., Gilman, M., Wiggs, J., and De-by denaturing the plasmid, annealing the mutagenicvera, A. (1983) Isolation of bacterial and bacteriophage RNAprimers, and repairing the plasmid with T4 DNA poly- polymerases and their use in synthesis of RNA in vitro. Methods

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P. D., Ed.) pp. 87–108, Academic Press, New York.transformation, the functional integrity of the T74. Davanloo, P., Rosenberg, A. H., Dunn, J. J., and Studier, F. W.RNAP may conveniently be monitored by assessing the

(1984) Cloning and expression of the gene for bacteriophage T7level of chloramphenicol resistance in vivo (12). ThisRNA polymerase. Proc. Natl. Acad. Sci. USA 81, 2035–2039.method of mutagenesis has been found to be useful not

5. King, G. C., Martin, C. T., Pham, T. T., and Coleman, J. E. (1986)only in the construction of point mutants (in whichTranscription by T7 RNA polymerase is not zinc-dependent and

one or a few base pairs were changed) but also in the is abolished by amidomethylation of cysteine-347. Biochemistryconstruction of insertion or deletion mutants involving 25, 36–40.as many as 6 base pairs. 6. Ikeda, R. A., and Richardson, C. C. (1987) Enzymatic properties

of a proteolytically nicked RNA polymerase of bacteriophage T7.Due to a concern that poorly regulated expression ofJ. Biol. Chem. 262, 3790–3799.the RNAP gene in pBH116 might lead to instability of

7. Grodberg, J., and Dunn, J. J. (1988) ompT encodes the Esche-the plasmid or might decrease the yield of RNAP, werichia coli outer membrane protease that cleaves T7 RNA poly-have used a compatible plasmid, pDM1.1 (21), whichmerase during purification. J. Bacteriol. 170, 1245–1253.provides a source of the lac repressor in the form of a

8. Jorgensen, E. D., Durbin, R. K., Risman, S. S., and McAllister,lac Iq gene located on a p15A replicon, to provide tight W. T. (1991) Specific contacts between the bacteriophage T3, T7,regulation. and SP6 RNA polymerases and their promoters. J. Biol. Chem.

266, 645–651.9. Zawadzki, V., and Gross, H. J. (1991) Rapid and simple purifica-CONCLUSION

tion of T7 RNA polymerase. Nucleic Acids Res. 19, 1948.In this work, we describe the construction of vectors

10. Gentz, R., Chen, C-H., and Rosen, C. A. (1989) Bioassay forthat allow the overexpression and rapid purification trans-activation using purified human immunodeficiency virusof histidine-tagged versions of T7, T3, and SP6 RNA that tat-encoded protein: Trans-activation requires mRNA syn-

thesis. Proc. Natl. Acad. Sci. USA 86, 821–824.polymerases. The enzymes appear to be identical toconventional RNAPs in all biochemical assays, and to 11. Van Dyke, M. W., Sirito, M., and Sawadogo, M. (1992) Single-



step purification of bacterially expressed polypeptides containing 19. Diaz, G. A., McAllister, W. T., and Durbin, R. K. (1996) The sta-bility of abortively cycling T7 RNA polymerase complexes is de-an oligo-histidine domain. Gene 111, 99–104.pendent upon template conformation. Biochemistry 35, 10837–12. Dunn, J. J., Krippl, B., Bernstein, K. E., Westphal, H., and10843.Studier, F. W. (1988) Targeting bacteriophage T7 RNA polymer-

ase to the mammalian cell nucleus. Gene 68, 259–266. 20. Muller, D. K., Martin, C. T., and Coleman, J. E. (1988) Process-13. Simon, M. A., and Studier, F. W. (1973) Physical mapping of the ivity of proteolytically modified forms of T7 RNA polymerase.

early region of bacteriophage T7 DNA. J. Mol. Biol. 79, 249– Biochemistry 27, 5763–5771.265. 21. Lanzer, M., and Bujard, H. (1988) Promoters largely determine

14. Sousa, R., Chung, Y. J., Rose, J. P., and Wang, B. C. (1993) The the efficiency of repressor action. Proc. Natl. Acad. Sci. USA 85,crystal structure of bacteriophage T7 RNA polymerase at 3.3 8973–8977.angstrom resolution. Nature 364, 593–599.

22. Studier, F. W., Rosenberg, A. H., Dunn, J. J., and Dubendorff,15. Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) ‘‘Molecular J. W. (1990) Use of T7 RNA polymerase to direct expression of

Cloning: A Laboratory Manual,’’ 2nd ed., Cold Spring Harbor cloned genes. Methods Enzymol. 185, 60–89.Laboratory, Cold Spring Harbor, NY.

23. Morris, C. E., Klement, J. F., and McAllister, W. T. (1986) Clon-16. Maslak, M., and Martin, C. T. (1994) Effects of solution condi-ing and expression of the bacteriophage T3 RNA polymerasetions on the steady-state kinetics of initiation of transcriptiongene. Gene 41, 193–200.by T7 RNA polymerase. Biochemistry 33, 6918–6924.

24. MacDonald, L. E. (1993) Termination by T7 RNA polymerase.17. MacDonald, L. E., Zhou, Y., and McAllister, W. T. (1993) Termi-in Ph.D. Thesis, State University of New York, Health Sciencenation and slippage by bacteriophage T7 RNA polymerase. J.Center at Brooklyn, Brooklyn, NY.Mol. Biol. 232, 1030–1047.

18. Martin, C. T., and Coleman, J. E. (1987) Kinetic analysis of T7 25. Martin, C. T., Muller, D. K., and Coleman, J. E. (1988) Process-ivity in early stages of transcription by T7 RNA polymerase.RNA polymerase-promoter interactions with small synthetic

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