Gene, 127 (1993) 99-103

0 1993 Elsevier Science Publishers

B.V. All rights reserved.

99

0378-l 119/93/$06.00

GENE 06994

A new cloning vector and expression strategy for genes encoding proteins
toxic to Escherichia coli
(Saccharomyces cereuisiae; POL3 gene; DNA polymerase 6; bacteriophage

T7)

William Clay Brown and Judith L. Campbell

Braun Laboratories, Division of Chemistry, California Institute of Technology, Pasadena, CA 91125, USA
Received by G. Wilcox: 20 April 1992; Revised/Accepted:

21 September/4

November

1992; Received at publishers:

14 December

1992

SUMMARY

Here, we describe a modification of a plasmid, pT7-7 [Tabor and Richardson, Proc. Natl. Acad. Sci. USA 262 (1985)
1074-10781, that allows expression of inserted genes from the phage T7 RNA polymerase promoter. The modification
is designed to suppress readthrough transcription from cryptic promoters and start points on the plasmid, in order to
reduce expression in the absence of T7 RNA polymerase and thus improve the vector for use in the expression of highly
toxic gene products. This vector (pT7SC) was used to stably clone the POL3 gene (encoding DNA polymerase 6) of
Saccharomyces cereuisiae, which destabilizes all other cloning and expression vectors tested. Previously described expression strategies proved ineffective in overexpressing the POL3 gene. A new strategy was developed which relies on
induction by infection with mutant T7 phage. This system efficiently overproduced the POL3 gene product.

INTRODUCTION

Since the cloning of DNA has become a fairly routine

procedure, the use of bacteria as a means of expressing
the cloned genes has become common. Very few failures
were encountered when the genes used were from Escherichia coli or other prokaryotic organisms, but as more
and more eukaryotic genes have been cloned and characterized a problem has arisen in their expression in bacteria. Often the gene product is toxic, as evidenced initially
by instability of the plasmid containing the gene. As a
means of countering this problem, expression vectors
Correspondence to: Dr. J.L. Campbell,
istry, 147-75 Caltech,
Fax (818) 449-0756.
Abbreviations:

Pasadena,

A, absorbance

Divisions

of Biology and Chem-

CA 91125, USA. Tel. (818)356-6053;

(1 cm); Ap, ampicillin;

B-lactamase (Bla); bp, base pair(s); A, deletion;

virus; IPTG, isopropyl-B-o-thiogalactopyranoside;

bla, gene encoding

HSV, herpes simplex

kb, kilobase
or

1000 bp; moi, multiplicity

of infection; nt, nucleotide(s);
ORF, open
reading frame; PA, polyacrylamide;
POW, gene encoding yeast DNA
polymerase 6; PolIk, Klenow (large) fragment of E. co/i DNA polymerase I; S., Saccharomyces;
SDS, sodium dodecyl sulfate.

were developed that rely on a bacteriophage T7 promoter, which should not be recognized by the RNA polymerases of E. co/i, to ensure that the expression of the
gene is tightly regulated (Tabor and Richardson, 1985;
Studier and Moffat, 1986). The T7 RNA polymerase may
be delivered to the system in a number of ways. A copy
of the T7 RNA polymerase gene may be carried on
another plasmid under the control of a thermolabile
phage h repressor and induced by temporarily raising the
temperature (Tabor and Richardson, 1985). Because
repression is not complete, however, genes are often
unstable even in these systems. Another method uses a h
lysogen cell line in which the T7 RNA polymerase gene
is under the control of the IPTG-inducible lac promoter.
To make the control of RNA polymerase gene expression
more stringent a plasmid containing the gene for lysozyme is present (Studier and Moffat, 1986). T7 lysozyme
specifically cleaves the residual RNA polymerase that is
produced prior to induction. When IPTG is added, the
cell is induced to produce much higher levels of T7 RNA
polymerase which rapidly outstrips the lysozyme activity
and goes on to transcribe the gene of interest. The final

100
method routinely used consists of infecting the cells containing the gene of interest with an Ml3 phage that carries the T7 RNA polymerase gene, also under EGCcontrol,
and inducing with IPTG. These latter systems have not
been universally effective in allowing for overexpression
of eukaryotic genes in bacteria. Often the yield of protein
is quite low, if the protein is produced at all.
Three nuclear RNA polymerases - a, 6 and a have
been characte~zed and shown to be encoded by essential
genes in Saccharomyces cerevisiae. Previous work has
shown that DNA polymerase 6 was encoded by gene
CffC2 (Sitney et al., 1989; Boulet et al., 1989), which has
since been renamed POL3. We and others have observed
that POL3-expressing plasmids are unstable in E. coEi
(Simon et al., 1991; P. Burgers, per. comm.) Observations
made while handling the gene suggested that uncontrolled expression, even at low levels, is responsible for
the instability, thus DNA polymerase 6 must be toxic to
E. co&. The goal of the present study is the development
of a vector and expression strategy that allows stable
cloning and expression of genes such as PUL3 that are
toxic to E. coii,

EXPERI~E~AL

AND DISCUSSION

pT7 SC
4121 bp

Fig. 1. Construction of pT7SC. Methods: Plasmid pKK223-3 (Pharmacia) was digested with Hind111+ SC&. The 82%bp fragment containing the bla gene upstream sequences and the rsnE terminators was
purified from a low-melting-agarose gel by phenol extraction (Perbal,
1984). The HindtII overhang was filled-in with PolIk to yield a bhmtended molecule. Piasmid pT7-7 was cut with EgJIXand the overhangs
were filled in. The 828-bp fragment was then blunt-end ligated into
pT7-7,Or~entation was determined by cutting recombinants with &WI.
Plasmid containing the properly oriented fra~ent was propagated and
then digested with CJaI. The overhangs were filled in and the 828-bp
fragment was inserted by blunt-end ligation. Orientation was again
checked by digestion with LtraI. The DNA replication origin fori) was
from ColEI.

(a) Construction of a vector which suppresses readthrough tran~ription

To stabilize the POL3 gene of S. cereu&siae,a new vector
was developed, based on those of Tabor and Richardson
(1985). The bia promoter and the Rho-independent transcription terminators 7; and TZfrom the rrnB operon of
E. co& (Brosius, 1984a) were cloned into pT7-7 (Tabor
and Richardson, 1985) upstream from the T7 promoter
(Fig. 1). The promoter was directed away from the cloning region and was followed downstream by T, and TZ
which occur in the inverted orientation from that found
in vivo but are still functional, though with decreased
efficiency (Brosius, 1984a). Some genes are so unstable in
bacterial vectors that there is difficulty in obtaining DNA
appropriate
for further characterization,
especially
sequence analysis. In order to allow for the cloning of
toxic genes with unknown sequence and orientation, the
extra promoter and terminator elements were also
inserted into the last restriction site of the pT7-7 polylinker, again such that the promoter is directed away from
the cloning region. The completed plasmid is shown in
Fig. 1 and has been named pT7SC (for stringent control).

to give rise to a 124-kDa protein (Boulet et al., 1989).

Constructs of POL3 beginning at either the A4iuI site or
the N&I site and running to the 3 HindIII site were
unstable in expression vectors pT7-7, pET3C (Studier
and Moffat, 1986) and pKK223-3 (Amman et al., 1983;
Brosius, 1984b). Constructs truncated to the &rr~I site
were stable in pT7-7 but protein was not observed upon
induction. This restriction site occurs down-stream from
the putative exonuciease sequences in region IV (Wong
et al., 1989; Simon et al., 1991) and this may have a bearing on both the stability of this construct and the lack of
expressed protein. The entire POL3 gene, however, was
successfully and stably cloned into pT7SC. A diagram of
this construct is shown in Fig. 2B. Inappropriate expression is apparently suppressed by the presence of the promoters and terminator sequences, and the POL3-carrying
plasmid is stable in a variety of commonly used cell lines.
Another gene which had previously proven difficult to
work with due to toxicity, yeast ~SP6~, was also stabilized in this vector (Smiley et al., 1992), suggesting that
this vector may be generally useful,

(b) Cloning the POL 3 gene

A map of the POL3 gene of S. cerevisiae, which encodes
the catalytic subunit of DNA polymerase 6 is shown in
Fig. 2A. This gene has a 3279-bp ORF, which is predicted

(c) Expression of POL3 by infection with mutant T7

bacteriophage
The pT7SC/POL3 construct relies on a T7 promoter
for the expression of the POW gene, and therefore a cell

101
Source of I7 RNA Pal

_
Start: ATG

II

111 I

Stop: TAA

Consmed Regions

C124kDa

pT7 SCIPOL3
(- : uniduced, + : induced)

Fig. 2. Linear map of S. cereuisiae POU gene (A), cloned in the pT7SC
plasmid (B). (A) ORF is shown as an open box, non-coding sequences
are medium thickness and shaded. The blackened boxes in the ORF
correspond to the conserved regions associated with u-like DNA polymerases (Wong et al., 1988). (B) The POW gene was obtained from a
YCp50 construct (between the Sal1 and Hind111 sites) in which the
upstream MluI site of POL.? was inserted at the vector Sal1 site such
that each was regenerated (pBL304). Plasmid pBL304 was digested with
Sal1 + Hind111 and ligated into these same sites in pT7SC. The resultant
recombinant was then cut with XbaI+MluI. The ends were filled in
and the plasmid was re-circularized. This construct (B) was used for
cloned gene expression.

line, BL21 (Studier and Moffat, 1986), that contains a

chromosomal copy of the T7 RNA polymerase gene was
used for expressing POL3. This cell line carries the POL3
gene-containing plasmid without alteration, but the DNA
polymerase 6 is not detectable upon induction with IPTG
(Fig. 3). A second approach was to use a proteasedeficient cell line and to provide the T7 RNA polymerase
by infecting with mGPl-2 which is an Ml3 strain that
contains the gene for T7 RNA polymerase under lac control. Again DNA polymerase 6 was not detected upon
infection and induction with IPTG (Fig. 3).
The protein encoded by gene 4 of bacteriophage T7 is
toxic and very difficult to clone (S. Tabor, per. comm.)
but is stable in E. coli during infection by T7 phage.
Possible phage T7 activities involved in suppression of
cellular functions include that of gene 2 protein, which
binds to and inhibits E. coli RNA polymerase, and of

Fig. 3. Expression of POI.3 is dependent on infection by mutant T7

bacteriophage. Fresh overnight cultures are diluted lOO-fold in LB with
20 ug Ap/ml and incubated at 30C to an A,,, = 1. Induction is through
either the addition of IPTG to 0.4 mM, infection with mGPl-2 at an
moi of 5 with addition of IPTG or by infection with bacteriophage
T7356- at an moi of 50. Incubations are then continued at 30 C for 1
h (T7 infection) or 3 h (IPTG and mGPl-2 induction). A 1 ml aliquot
is removed from each sample, the cells are pelleted and then resuspended in 80 pl of cracking buffer (60 mM TrisHCl pH 6.8/l% mercaptoethanol/lO% glycerol/3% SDS/O.01% bromophenol blue). Half
of this volume is loaded onto a 0.1% SDS-7.5% PA gel. E. coli RNA
polymerase was run as molecular weight standard. The proteins are
visualized by staining with Coomassie blue. The source of the T7 RNA
polymerase is indicated above the lanes. Cells are E. coli HMS 174
containing either vector or vector with the POL3 gene and are either
uninduced (-) or induced (+). Only the lane containing cells harboring
POW plasmid that are infected with bacteriophage T7356- displays
production of a 124-kDa protein (arrow) corresponding to DNA polymerase 8, the POW product.

gene 0.7, which encodes a protein kinase that also inactivates the host RNA polymerase. There are several genes
in the early expressed portion of the genome that encode
rather small proteins that have been detected upon infection but whose activities have not been characterized.
These proteins may be peptide inhibitors of proteases or
other metabolic enzymes. We reasoned that DNA polymerase 6 might be more compatible with the host during
infection by T7. A mutant phage that produces T7 RNA
polymerase but that does not progress through its life
cycle because of amber mutations in genes 3,5 and 6 was
used. Genes 3 and 6 encode very potent endo- and exonucleases which normally degrade the host DNA during
an infection. Gene 5 encodes the catalytic subunit of T7
DNA polymerase, which is essential for replicating the
phage genome. Upon infection with these mutant phage,
effective overproduction
of DNA polymerase 6 is
observed (Fig. 3). Within several minutes of infection

102
large amounts of DNA polymerase 6 are detectable. The
cells generally lyse between 150 and 180 min after infection. These experiments have been repeated a number of
times with all three systems and the results are invariant,
that is polymerase 6 is not produced using the traditional
systems but large amounts are obtained using phage
infection.
(d) Partially deleted and truncated constructs of DNA
polymerase 6
In order to carry out simple structure function studies,
several ~01338mutants were constructed. The stable construct of the full-length PUL3 gene was cut at restriction
sites tanking sequences predicted to encode exonuclease
function and religated (Fig. 4 B and D). The first internal
deletion, formed by EcoRV digestion, removes a 252bp
segment which contains the exoI, exo1 and exoI1
sequences. Site-specific mutations in these sequences in
vivo resulted in the apparent reduction of proofreading
activity of polymerase 6 (Simon et al., 1991). Ligation

HindIll
product

= 124 ku

Stan: ATG

Stop: TAA

Iindfli
Produa

= 114kDa

regenerates a single EcoRV site which can be used to

ensure the gene is in frame. The other internal deletion
retains the first nine triplets at the N terminus but
removes the next 1212 bp to the second EcoRV site, combining the effects of the first deletion and the following
truncation. An N-terminally truncated form of the gene
was also cloned into the Ndel site of pT7SC as shown in
Fig. 5C. The N-terminal truncation beginning at the NldeI
site removes 660 bp from the N terminus, while retaining
all of the six conserved regions. This construct differs
from the others in that the vector ribosome binding site
and ATG spacing are retained (see position of XbaI and
NdeI sites in Fig. 1).
In a number of trials both the full-length (124 kDa)
and truncated (98 kDa) versions of the protein are overexpressed. When bacteriophage infection is accompanied
by over-production
of protein, the protein profile
observed is radically altered as evidenced by the lanes of
124-kDa and 98-kDa induction in Fig. 5, a representative
SDS-PA gel of these experiments. The internal deletions
(114 and 78 kDa) did not give rise to protein upon induction (data not shown).
Site-specific mutants of PUL3 affecting the deleted exonuclease region showed DNA polymerase activity in vitro
and complemented ~013 mutants (Simon et al., 1991).
However, another study of site specific mutants of the
homologous regions in HSV Pol (Gibbs et al., 1991)
showed some mutants completely lacked polyme~zation
activity. These authors suggested that the exonuclease
region does not fold as a separate domain but interacts
with the polymerase active site. If this is the case then
deleting these regions may destabilize the protein and this

HindIII

HindIII
Ndel

EmRV
EcoRV

Product

MI11

= 9% ma

Fig. 4. Deletion and truncation constructs of PUL3. The thin single

lines represent removed sequences, medium shaded boxes are noncoding regions and the large open boxes are the ORFs with the six conserved regions indicated in black. The full-length construct is shown
(A) as it appears in pT7SCjPOt3. This was digested with EcoRV and
re-circularized (B). The resulting plasmid would give rise to a 1ICkDa
protein. A truncated form of POL.3 (C) was constructed by digesting
both pBL304 and pT7SC with N&I +HindIII. The digests were then
mixed for ligation. Re~rnbin~~
were screened first by size and then
restriction mapping to identify the proper construct which should yield
a 9%kDa protein. A larger deletion (D) was constructed by digesting
pT7SC/POW with NcoI + EcoRV. The ends were filled in and the plasmid was re-circularized. This should give rise to a 78-kDa protein.

N-

-98

Fig. 5. Synthesis of various forms of DNA polymerase S by infection

with bacte~ophage T7356-. Cell lines contained the constructs indicated
by molecular sizes (in kDa) and are either infected (+) or uninfected (-).
Expression is detected only for the full-length (124 kDa) and truncated
(98 kDa) constructs. The samples and gel were handled as described in
the legend to Fig. 3.

103
is why expression from the 11CkDa and 7%kDa constructs is not detected. In keeping with this interpretation,
as stated above in section (b), a construct deleting the
entire N terminus through the exonuclease domain (BsmI
site) could be stably cloned in any vector, though the
protein is not detected upon induction.

Louis) for providing pBL304, and Dr. Louis J. Roman0

(Wayne State University, Detroit) for providing T7356phage. This work was supported by NIHGMS 25508 and
NRSA CA08692 to W.C.B.

REFERENCES

(e) Conclusions
The vector presented here, pT7SC, successfully suppresses the read-through transcription of the toxic POL3
gene of S. cereuisiae thus stabilizing it in E. coli. The
strategies most commonly used for expression of genes
in T7-based vectors were not effective in overexpressing
the POL3 gene. Infection with mutant T7 bacteriophage
led to high levels of overexpression. We have obtained
up to 15 mg of protein from as little as 3 grams of cells.
The protein is not found in the soluble cell extract but is
included and found in the insoluble material. Purification
of active protein from the inclusion bodies will be published elsewhere (Brown et al., 1993).
There are many eukaryotic and also prokaryotic proteins that have potential clinical or industrial use but are
not abundant. The ability to express them in bacteria
would provide a large and readily available supply of
such proteins. While initial gene isolation and renaturation of overexpressed proteins have become common,
a problem remains in inducing the cells harboring the
gene of interest to produce protein, when the product is
for some reason incompatible with the host. The strategy
we describe is a way of removing cellular control and
directing the cell to produce these proteins in abundance.
We are currently investigating the possibility of placing
the early gene region of T7 on a plasmid to reduce the
problem of cell lysis associated with phage infection.

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