Biotechnol. Appl. Biochem.

(1999) 30, 1925 (Printed in Great Britain) 19

Purication and properties of Thermus liformis DNA
polymerase expressed in Escherichia coli
Jeong Jin Choi, Seung Eun Jung, Hyun-Kyu Kim and Suk-Tae Kwon
1
Department of Genetic Engineering, Sungkyunkwan University, 300 Chunchon-Dong, Jangan-Ku, Suwon 440-746, Korea
The gene encoding Thermus liformis (T) DNA poly-
merase was expressed under the control of the tac pro-
moter on a high-copy plasmid, pJR, in Escherichia coli.
The T DNA polymerase was puried by using heat
treatment and DEAE-Sephacel column chromato-
graphy. The puried enzyme had a molecular mass of
92 kDa, as estimated by SDS/PAGE. The optimum pH
and temperature of the enzyme were 8.49.0 and
7072.5 mC respectively. The half-life of the enzyme at
94 mC was approx. 40 min. The enzyme was activated
by the bivalent cations, Mg
2
+
andMn
2
+
, andwas inhibited
by EDTA. The optimal Mg
2
+
concentration of the
enzyme was 4 mM. The optimal conditions for the PCR
reaction were slightly different from those for the
enzyme activity except for the optimal Mg
2
+
concen-
tration. Low concentrations of KCl had no effect on
either the enzymic activity or the PCR amplication.
The result of the PCRexperiment withthe enzyme indi-
cates that T DNA polymerase might be useful in DNA
amplication.
Introduction
PCR has become a powerful method for the identication
and amplication of genes, for direct sequencing and for
clinical diagnosis. In particular the uses of PCR in clinical
diagnosis are broadening to include diagnoses of new
mutation and monogenic disease, the analysis of biological
evidence and the detection of oncogenes and human
infectious disease [1]. Early PCR experiments used the
thermolabile Klenow fragment, which had to be added to
every cycle [2]. The introduction of a thermostable DNA
polymerase allowed the automation of the process [3]. A
thermostable DNA polymerase was much more suitable for
the thermocycles during PCR [1,3]. Accordingly, a thermo-
stable DNA polymerase has become an indispensable
component of PCR technology.
Thermostable DNA polymerase was rst isolated from
the thermophilic bacterium Thermus aquaticus YT-1 (Taq
DNA polymerase) ; its properties were reported in [4,5].
Similar enzymes have been isolated from other Thermus
strains, including T. avus [6], T. thermophilus HB-8 [7] and
T. caldophilus GK24 (TcaDNA polymerase) [8]. Each enzyme
has slightly different characteristics; however, no informa-
tion is available on the properties of the thermostable DNA
polymerase from T. liformis (T DNA polymerase).
T. liformis was isolated from a New Zealand hot spring
and described as member of the genus Thermusby Hudson et
al. [9]. T. liformis always forms long laments consisting of
chains of cells and can thus be distinguished from other
Thermus strains in morphology.
The TDNApolymerase gene has recently been cloned
and sequenced [10]. The amino acid sequence of T DNA
polymerase was deduced from the nucleotide sequence of
the cloned gene. T DNA polymerase is composed of 833
amino acid residues and has a molecular mass of 93890 Da.
The deduced amino acid sequence of T DNA polymerase
showed a high sequence similarity to Escherichia coli DNA
polymerase I-like DNA polymerases.
Here we report (1) the expression of the gene for T
DNA polymerase, (2) the purication and properties of
T DNA polymerase and (3) the optimal conditions for
PCR with T DNA polymerase.
Materials and methods
Bacterial strains and culture conditions
T. liformis (ATCC no. 43280) [9] was prepared as described
by Ramaley and Hixson [11]. E. coli strain MV1184 [12] was
used as the host for plasmid preparations. The E. coli cells
were grown in LuriaBertani medium at 37 mC. The plate
was solidied with 1.5% (w\v) agar; ampicillin (100 g\ml)
was added when needed.
Molecular cloning of the gene for T DNA
polymerase
Most of the methods used for molecular cloning were based
on those of Sambrook et al. [12]. Genomic DNA of
T. liformis and plasmid DNA were isolated by a method
modied from that of Marmur [13] and a modied alkaline
lysis method [12] respectively. A 2.5 kb fragment containing
the whole T DNA polymerase gene was prepared by PCR
Abbreviations used: DTT, dithiothreitol ; Taq DNA polymerase, DNA
polymerase isolated from Thermus aquaticus YT-1; Tca DNA polymerase,
DNA polymerase isolated from Thermus caldophilus GK24; Tfi DNA
polymerase, DNA polymerase isolated from Thermus filiformis.
1
To whom correspondence should be addressed.
# 1999 Portland Press Ltd
20 J. J. Choi and others
Figure 1 Construction of the recombinant plasmid pTFP11
The Tfi DNA polymerase gene, amplified by PCR with the T. filiformis genomic
DNA as a template DNA, was introduced into an expression vector pJR by
using the EcoRI and SalI sites. Abbreviations : E, EcoRI ; S, SalI.
amplication with the T. liformis genomic DNA and primers
that annealed to opposite ends of the coding region, on the
basis of the published sequence of the gene [10]. The 5h (N-
terminal) primer sequence was 5h-NNNNGAATTCNATG-
ACCCCACTTTTTGACC-3h, and the 3h (C-terminal)
primer sequence was 5h-NNNNGTCGACTCAATCC-
TGCTTCGC-3h, which created the underlined unique EcoRI
and SalI restriction enzyme sites respectively, at each end
of the amplied DNA fragment. The amplied product by
PCR was digested with EcoRI and SalI ; the 2.5 kb DNA
fragment was isolated from the low-melting-temperature
agarose gel. The fragment was ligated into an expression
vector pJR [14] that had been digested with EcoRI and SalI,
giving a fusion that used the tac promoter, the ribosome-
binding site and a transcription terminator from the vector.
The ligate was transformed into E. coli MV1184 as described
by Hanahan [15]. The correct construction of the expression
vector for the T DNA polymerase gene was identied by
restriction enzyme analysis of plasmid minipreps. The
resulting recombinant plasmid pTFP11 (Figure 1) possessed
11 bp between the ShineDalgarno sequence [16] and an
ATG codon.
T DNA polymerase activity assay
The basic reaction mixture (50 l) contained 25 mM Tris\
HCl, pH 8.5, 40 mM KCl, 2 mM MgCl
2
, 1 mM 2-mercapto-
ethanol, 100 MdATP, 100 MdCTP, 100 MdGTP, 10 M
dTTP including 0.5 Ci [methyl-1h,2h-
3
H]thymidine 5h-tri-
phosphate, 1.25 g of activated calf thymus DNA, and
enzyme solution. The mixture was incubated for 10 min at
75 mC [8]. The mixture was spotted on a DE-81 lter paper
disk (23 mm), dried on a heat block, then washed with 0.5 M
Na
2
HPO
4
, pH 7.0, for 10 min. This was followed by a wash
with 70% (v\v) ethanol for 5 min. A dry lter paper disk was
used to measure the amount of [
3
H]dTTP incorporation
with a Beckman LS 6500 scintillation system. One unit of
enzyme is dened as 1 pmol of product synthesized in
10 min at 75 mC.
Purication of the expressed T DNA polymerase in
E. coli
A 40 ml sample of an overnight culture of E. coli MV1184
harbouring the recombinant plasmid pTFP11 grown in
LuriaBertani broth containing ampicillin was transferred to
4 litres of the same medium, and the culture was incubated
at 37 mC. When the A
600
of the culture had reached approx.
0.6, the culture was induced by the addition of isopropyl -
D-thiogalactoside at a nal concentration of 0.2 mM, then
incubated at 37 mC for 6 h. The cells were harvested by
centrifugation. The cell pellet was resuspended in buffer A
[10 mM Tris\HCl (pH 8.0)\1 mM MgCl
2
] containing 1 mM
dithiothreitol (DTT) and 1 mM PMSF, then disrupted by
sonication. The sonicated cells were centrifuged at 10000 g
for 20 min to remove E. coli cell debris. The nucleic acids in
the supernatant were precipitated for 30 min by 1% (w\v)
streptomycin sulphate at room temperature, then removed
by centrifugation. The supernatant was incubated in a water
bath at 80 mC for 30 min to denature E. coli proteins. After
removal of the denatured proteins by centrifugation at
10000 g for 20 min, the supernatant was dialysed against
buffer A, then applied to a DEAE-Sephacel2 column that had
been equilibrated with buffer A. The elution of adsorbed
protein was performed with a linear gradient of KCl
(00.5 M) in buffer A. The fractions showing DNA poly-
merase activity were pooled and dialysed against buffer A.
The puried T DNA polymerase was then analysed and
assayed.
Protein determination and electrophoretic analysis
Protein concentration was determined by the procedure of
Lowry et al. [17], with BSA as a standard. SDS\PAGE was
performed by the method of Laemmli [18] with 7% (w\v)
polyacrylamide.
# 1999 Portland Press Ltd
Properties of Thermus liformis DNA polymerase 21
Primers and PCR conditions for searching optimal
condition of PCR
Oligodeoxynucleotide primers that annealed to -phage
DNA [19] were designed to give 2, 4 and 6 kb amplied
DNAfragments by PCR. The sequences of the primers were
as follows: primer 1 (20012020), 5h-CGCCACGACGAT-
GAACAGAC-3h (forward) ; primer 2 (39814000), 5h-
CACTGGCCCGTGCCGTGGAG-3h (reverse) ; primer 3
(59806000), 5h-TCCTCATAACGGAACGTGCCG-3h (re-
verse) ; primer 4 (79928013), 5h-GGTTTTCAACGGCC-
TGCTCAAG-3h (reverse). The numbers in parentheses
indicate the position of the -phage DNA.
PCR mixture (50 l) contained 2.5 pmol of primers,
each dNTP at 200 M, 1 unit of TDNA polymerase, and -
phage DNA as a template DNA. After a single 5 min
denaturation step at 95 mC, PCR (25 cycles) was done by
1 min of denaturation at 94 mC, 1 min of annealing at 64 mC,
and 1 min of extension at 72 mC. A nal 5 min extension at
72 mC was performed before terminating the reaction.
Results and discussion
Purication of T DNA polymerase
The merits of the production of thermostable enzymes in
the E. coli system include not only the production of the
enzyme in large quantity but also the ease of purication by
using different physical properties of the thermostable
enzymes compared with those of the host proteins. These
advantages were applied to the purication of T DNA
polymerase in E. coli.
To measure the expression level, E. coli MV1184 har-
bouring pTFP11 was cultured in the presence of isopropyl -
D-thiogalactoside. The expression level of T DNA poly-
merase reached 52 units\ml, roughly a 43-fold increase
compared with production by T. liformis. The large scale
culture of E. coli MV1184 harbouring pTFP11 was done in a
4 litre fermentor. At rst the harvested cells (39.2 g) were
sonicated and treated with streptomycin sulphate to remove
nucleic acids. The T DNA polymerase was then puried by
heat treatment and DEAE-Sephacel column chromato-
graphy. The purication of the enzyme is summarized in
Table 1. The specic activity of the puried enzyme was
more than 28.8-fold that of the sonicated extract ; recovery
Table 1 Purication summary of recombinant T DNA polymerase
expressed in E. coli
The purification was started with 39.2 g wet weight of cells.
Purification
Total
protein
(mg)
Total
activity
(k-units)
Specific
activity
(units/mg)
Recovery
(%)
Sonicated extract 2480.0 219.4 88.5 100
Heat treatment 63.4 72.29 1140.3 33.0
DEAE-Sephacel 19.2 49.02 2552.9 22.3
Figure 2 SDS\PAGE analysis of T DNA polymerase
Electrophoresis was performed on a vertical gel containing 7% (w/v)
polyacrylamide with a Mighty Small Kit II system (Hoffer Scientific Instruments).
Lane 1, uninduced cell ; lane 2, sonicated extract ; lane 3, heat treatmemt ; lane
4, DEAE-Sephacel column chromatography; lane M, low-molecular-mass
markers (molecular masses are indicated at the right).
Figure 3 Effect of pH on the activity of T DNA polymerase
The reaction was performed at 75 mCin the following buffers at a concentration
of 50 mM in the presence of 2 mM MgCl
2
: Tris/HCl (#), glycine/NaOH ($).
The pH values shown are those at 25 mC.
was approx. 22.3% on the basis of the total activity of the
sonicated extract. The purication of the enzyme was
monitored by SDS\PAGE (Figure 2). Most proteins orig-
inating from E. coli were almost removed by heat treatment ;
nucleic acids and trace proteins were completely removed
by DEAE-Sephacel column chromatography. The puried
sample of T DNA polymerase seemed to be homogeneous
by SDS\PAGE. A single protein band corresponding to a
molecular mass of 92000 Da was obtained by SDS\PAGE.
This molecular mass showed good agreement with
93890 Da calculated from the deduced amino acid sequence
[10].
# 1999 Portland Press Ltd
22 J. J. Choi and others
Figure 4 Effect of temperature on the activity of T DNA polymerase
Enzyme activity was assayed at the indicated temperatures in 25 mM Tris/HCl,
pH 8.5, containing 2 mM MgCl
2
.
Figure 5 Thermostability of T DNA polymerase
Tfi DNA polymerase was dissolved at a concentration of 10 g/ml protein in
10 mM Tris/HCl, pH 8.5, containing 2 mM MgCl
2
and 0.01% BSA. The
enzyme solutions, in the absence of template DNA, were incubated in sealed
Eppendorf tubes for 02 h at 75 mC (#) and 94 mC ($). After incubation for
the indicated durations, the residual activity was measured as described in the
Materials and methods section.
Properties of the puried T DNA polymerase
Effect of pH on the activity and stability of T DNA polymerase
The dependence of T DNA polymerase activity on pH was
determined in the pH range 7.610.6. The buffers used were
50 mM Tris\HCl (pH 7.69.4) and 50 mM glycine\NaOH
(pH 9.010.6). The results are shown in Figure 3. The
enzyme has an optimum catalytic activity at alkaline pH
values. From pH 7.6 its activity increases slowly, reaching a
maximum at pH 8.49.0 and declining sharply at pH 9.6.
These results are similar to the pH dependence of TaqDNA
polymerase activity [5]. The pH stability of the enzyme was
Figure 6 Effect of KCl on the activity of T DNA polymerase
Enzyme activity was assayed separately under the basic reaction conditions with
various concentrations of KCl.
examined by incubating the enzyme solution at different pH
values for 36 h at 25 mC; the residual activity was then
measured under the basic reaction conditions. The results
indicated that T DNA polymerase is relatively stable at
pH 7.810.6 (results not shown).
Effect of temperature on the activity and stability of T DNA
polymerase The effect of temperature on the T DNA
polymerase activity was determined in the range 5585 mC.
The optimumtemperature was 7072.5 mC(Figure 4). Figure
5 shows the thermostability of the enzyme at 75 and 94 mC.
The enzyme was fairly stable at 75 mC; however, at
temperatures above 90 mCthe thermostability of the enzyme
decreases drastically. The half-life at 94 mCin the presence of
0.01% BSA was approx. 40 min.
Effect of KCl on the activity of TDNA polymerase The results
of a study on the inuence of KCl concentration on the T
DNA polymerase activity are shown in Figure 6. Its optimal
concentration is broad (050 mM), declining sharply at
100 mMor higher. There is no stimulation of enzyme activity
in the 150 mM range. In contrast, Taq DNA polymerase is
stimulated 2-fold by the addition of KCl [5], and its optimal
KCl concentration is 100200 mM. Therefore optimal
results are obtained at lower concentrations of KCl with T
DNA polymerase than with Taq DNA polymerase.
Effects of bivalent cations, EDTA and DTTon the activity of T
DNA polymerase Figure 7 shows the effects of different
concentrations of Mg
2
+
and Mn
2
+
on the activity of T DNA
polymerase. The optimal Mg
2
+
concentration is 4 mM and
the optimal Mn
2
+
concentration is 1 mM. Comparing the
inuences of the two bivalent cations, we found that TDNA
# 1999 Portland Press Ltd
Properties of Thermus liformis DNA polymerase 23
Figure 7 Effects of bivalent cations, Mg
2
+
and Mn
2
+
, on the activity of T
DNA polymerase
Enzyme activity was assayed separately under the basic reaction conditions with
various concentrations of Mg
2
+
(#) or Mn
2
+
($).
Figure 8 Effect of pH on PCR amplication with T DNA polymerase
The amplification of -phage DNA fragment was performed in a 50 l reaction
mixture containing 50 mM Tris/HCl (A) or glycine/NaOH (B) and 1.2 mM
MgCl
2
at the pH values indicated. PCR was performed as described in the
Materials and methods section. A 5 l sample of each mixture was subjected to
electrophoresis on 0.8% agarose gel and stained with ethidium bromide. The
positions of size markers are indicated at the left.
polymerase exhibited 3-fold higher activity in the presence
of Mg
2
+
than Mn
2
+
. The effects of EDTA (05 mM) and DTT
(050 mM) on the activity of the enzyme were examined by
assaying enzyme samples in the presence of EDTA and DTT
at various concentrations. TDNApolymerase was inhibited
by 1.5 mMEDTA; however, DTThad no signicant inuence
on activity (results not shown). T DNA polymerase was
activated by bivalent cations and was inhibited by EDTA.
These results indicate that the highly conserved regions of
the polymerase domain of T DNA polymerase also contain
a few carboxylates [20], and that bivalent cations are used
as cofactor [21].
Figure 9 Effect of Mg
2
+
on PCR amplication with T DNA polymerase
The amplification of -phage DNA fragment was performed in a 50 l reaction
mixture containing 50 mM Tris/HCl, pH 9.0, and concentrations of MgCl
2
as
indicated. PCR was performed as described in the Materials and methods
section. A sample 5 l of each mixture was subjected to electrophoresis on
0.8% agarose gel and stained with ethidium bromide. The positions of size
markers are indicated at the left.
Figure 10 Effect of KCl on PCR amplication with T DNA polymerase
The amplification of -phage DNA fragment was performed in a 50 l reaction
mixture containing 50 mM Tris/HCl, pH 9.0, and 1.5 mM MgCl
2
with concen-
trations of KCl as indicated. PCR was performed as described in the Materials
and methods section. A 5 l sample of each mixture were subjected to
electrophoresis on 0.8% agarose gel and stained with ethidium bromide. The
positions of size markers are indicated at the left.
Optimal conditions for PCR using T DNA
polymerase
DNA polymerases from the genus Thermus are thermo-
stable, so it is not necessary to replenish the enzyme after
each PCR cycle as with E. coli DNA polymerase I. For PCR it
is important to know the optimal conditions when using this
enzyme.
To nd the optimum pH, -phage DNA, with a pH
range of 7.69.8, was amplied in the presence of T DNA
polymerase by using both primers 1 and 2. The buffers used
were 50mM Tris\HCl (pH 7.69.4) and 50 mM glycine\
NaOH (pH 9.09.8). The results are shown in Figure 8. The
-phage DNA fragment was well amplied at pH 8.09.4
# 1999 Portland Press Ltd
24 J. J. Choi and others
Figure 11 Analysis of PCR products for optimal PCR conditions
The amplification of -phage DNA fragment was performed in a 50 l reaction
mixture containing 50 mM Tris/HCl, pH 9.0, and 1.5 mM MgCl
2
. PCR was
performed as described in the Materials and methods section. A 5 l sample of
each mixture was subjected to electrophoresis on 0.8% agarose gel and
stained with ethidium bromide. Lane M, DNA molecular size markers (1 kb
DNAladder ; sizes are shown at the left) ; lane 1, the product size expected with
primers 1 and 2 was 2 kb; lane 2, the product size expected with primers 1 and
3 was 4 kb; and lane 3, the product size expected with primers 1 and 4 was
6 kb.
in 50 mM Tris\HCl and at pH 9.09.4 in 50 mM glycine\
NaOH. However, amplication in glycine\NaOH was lower
than for the same pH range in Tris\HCl. For PCR with T
DNA polymerase the optimum pH range is 8.89.2.
To nd the optimal concentration of Mg
2
+
, -phage
DNA, with a concentration of Mg
2
+
in the range 0.7510 mM,
was amplied by using both primers 1 and 2. The results are
shown in Figure 9. The optimal Mg
2
+
concentration for PCR
is 1.5 mM; there was no amplication below 0.75 mM. The
results indicate that the optimal Mg
2
+
concentration for PCR
is slightly different from that of enzyme activity, whose
optimal Mg
2
+
concentration is 4 mM(see Figure 7). However,
-phage DNA fragment was not amplied in the presence of
Mn
2
+
instead of Mg
2
+
.
There was no effect of low concentrations of KCl on
PCR catalysed by T DNA polymerase; above 80 mM KCl,
amplication was inhibited (Figure 10). This is consistent
with the effect of KCl on enzyme activity (see Figure 6). This
is again in contrast with the behaviours of Taq DNA
polymerase ([22], and S.-T. Kwon, unpublished work) and
DNA polymerase Tca ([8], and S.-T. Kwon, unpublished
work), where the addition of KCl is essential for PCR
amplication. With most other DNA polymerases from
the genus Thermus, KCl is an indispensable ingredient.
Therefore the T DNA polymerase could conceivably have
a unique utility in amplifying certain template DNA species
that cannot be amplied with KCl-requiring polymerase.
As seen from the above results, we found that the
optimal buffer for PCR with T DNA polymerase is 50 mM
Tris\HCl (pH 9.0)\1.5 mM MgCl
2
, with 0.01% BSA as a
stabilizer. -phage DNA fragments were amplied in the
presence of T DNA ploymerase, in conjunction with
primers 1 and 2, primers 1 and 3 and primers 1 and 4 under
the optimal conditions of PCR determined above. T DNA
polymerase readily produced 2, 4 and 6 kb DNA fragments
(Figure 11). This result indicates that the DNA amplication
capability of T DNA polymerase is superior and might be
useful in molecular biology, clinical diagnosis, and genetic and
evolutionary analyses.
As mentioned in the Introduction section, PCR has
already become a widespread research technique. Although
the results are good in many cases, some points should be
considered for exploration if better results are required or
if the reaction fails altogether [1]. In addition, in a recent
study [23] it was noted that the PCR-inhibiting effect of
various components in biological samples could be elimi-
nated to some extent by the use of the appropriate
thermostable DNA polymerase. T DNA polymerase has
some differences in properties compared with the DNA
polymerases from other thermophilic bacteria. There is
therefore the possibility that T DNA polymerase might be
useful for use on a PCR sample that cannot be amplied well
with other DNA polymerases; we are conducting experi-
ments to establish the PCR conditions for various samples.
Acknowledgment
This study was supported by the Academic Research Fund
(GE97-233) of the Ministry of Education, Republic of Korea.
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Received 25 January 1999\23 March 1999; accepted 13 April 1999
# 1999 Portland Press Ltd