J. Mol. BioE. (1991) 219, 37-44
Use of Bacteriophage T7 Lysozyme to Improve an Inducible T7 Expression System F. William Studier Biology Department Brookhaven National Laboratory Upton, NY 11973, U.S.A. (Received 9 August
1990; accepted 11 January
1991)
Bacteriophage T7 lysozyme, a natural inhibitor of T7 RNA polymerase, can reduce basal activity from an inducible gene for T7 RNA polymerase and allow relatively toxic genes to be established in the same cell under control of a T7 promoter. Low levels of T7 lysozyme supplied by plasmids pLysS or pLysL, which are compatible with the PET vectors for expressing genes from a T7 promoter, are sufficient to stabilize many target plasmids and yet allow high levels of target protein to be produced upon induction of T7 RNA polymerase. Higher levels of lysozyme supplied by plasmids pLysE or pLysH reduce the fully induced activity of T7 RNA polymerase such that induced cells can continue to grow Different configurations of the and produce innocuous target proteins indefinitely. expression system can maintain several different steady-state levels of target gene expression. The presence of T7 lysozyme has the further advantage of facilitating the lysis of cells in preparing extracts for purification of target gene products. Keywords: T7 lysozyme; T7 RNA polymerase; T7 expression system; toxic target genes; cloned genes
1. Introduction
the range and versatility expression system.
Bacteriophage T7 RNA polymerase actively and selectively directs transcription from a relatively large promoter that is unlikely to be found in DNA unrelated to T7 (Chamberlin et al., 1970). This selectivity provides the basis for gene expression systems in Escherichia coli (Tabor & Richardson, 1985; Studier & Moffatt, 1986; Rosenberg et al., 1987) and potentially in a variety of cell types (Fuerst et al., 1986; Chen et al., 1987; Dunn et al., 1988). Configurations where T7 RNA polymerase is inducible are convenient for expressing a wide variety of target genes. However, the polymerase is so active that even a small basal level in the uninduced cell can prevent relatively toxic target genes from being established or make them difficult to maintain. Furthermore, the induced level of transcription of target DNA becomes so high that, even if the target gene is innocuous, the cell eventually stops growing and dividing. T7 lysozyme is a bifunctional protein that cuts a bond in the cell wall of E. coli (Inouye et al., 1973) and selectively inhibits T7 RNA polymerase by binding to it, a feed-back mechanism that ensures a controlled burst of transcription during T7 infection (Moffatt & Studier, 1987). These activities of T7 lysozyme are now shown to be useful for increasing
of
an
inducible
T7
2. Materials and Methods (a) The T7 expression
system
T7 expression hosts were E. coli BL21(DE3) and HMS174(DE3), which are lysogens of DE3, a phage lambda derivative that carries the gene for T7 RNA polymerase under control of the ZacUVS promoter (Studier & Moffatt, 1986). Target genes were cloned under control of the strong $10 promoter for T7 RNA polymerase in plasmid PET-1, a derivative of plasmid pBR322 that confers resistance to ampicillin (Rosenberg et al., 1987). The 410 promoter in PET-1 is directed counterclockwise across the BamHI site of pBR322, opposed to the tet promoter. Plasmid PET-3 is similar to PET-1 except that it also carries the T$ transcription terminator located just past the BumHI site (Rosenberg et aZ., 1987). Conditions for growing cultures, inducing synthesis of T7 RNA polymerase and expressing target genes have been described (Studier & Moffatt, 1986) and here in the Figure legends. Ability of induced cells to form colonies was tested by adding uninduced cells to 2.5 ml of melted top agar containing 1 mM-IPTGt and 9.5 mg of ampicillin
t Abbreviation used: IPTG, isopropyl-B-othiogalactopyranoside. 37
00X-2836/91
/090037-08
$03.00/O
0
1991 Academic
Press Limited
38
F. W. Studier
and plating onto 20 ml of solidified lacked these additives.
bottom
agar that
(b) Tr lysozyme clones T7 gene 35, the gene for T7 lysozyme, was cloned in the BarnHI site of pACYC184 (Chang & Cohen, 1978; Rose, 1988), a plasmid that is compatible with pBR322-based plaamids such as the PET plasmids. A fragment from plasmid pAR.410 (Studier & Rosenberg, 1981: base-pairs 10,665 to 11,296 of T7 DNA) contains the coding sequence for the last 13 amino acids of T7 gene 3, the entire coding sequence of gene 3.5 (base-pairs 10,706 to 11,161), the 43.8 promoter for T7 RNA polymerase, and the coding sequence for the first 24 amino acids of gene 3.8 (Dunn & Studier, 1983). Plasmid pLysE (Expressed) has this fragment oriented so that lysozyme mRNA is transcribed from the tet promoter of pACYCl84; pLysS (Silent) has the fragment in the opposite orientation (Fig. 1). A shorter gene 3.5 fragment, which ends at base-pair 11,164 and therefore lacks the 43.X promoter and the beginning of gene 3.8, was also cloned. Plasmid pLysH (High level) has this shorter fragment oriented so that lysozyme mRNA is transcribed from the tet promoter; pLysL (Low level) has the fragment in the opposite orientation. These lysozyme plasmids confer resistance to chloramphenicol; cells containing them are typically grown in medium containing 10 to 25 pg chloramphenicol/ml or are selected on plates to which 250 to 625 pg of chloramphenicol has been added to the top agar. (c) Target
genes
Each of the genes of bacteriophage T7 (Dunn $ Studier. 1983) has been cloned individually under control of a class III T7 promoter (Studier & Rosenberg, 1981; and work to be described elsewhere). Genes naturally adjacent to a class III promoter were cloned along with their promoter in the BamHI site of pBR322; the other genes were cloned under control of the 410 promoter in the BarnHI site of PET-1. Some genes cloned in PET-1 were not separated from an adjacent class IT promoter and were therefore under control of both 410 and the class II promoter. A set of 54 target pl&smids, representing 53 different T7 genes (all except the genes for T7 RNA polymerase, T7 lysozyme and the T7 protein kinase) was used in this work. including a clone of genes I.1 and 1.2 under control of $10. a combination more toxic than either gene cloned individually. (d) Isolation
of plasmid
DNA
The following rapid and convenient procedure was used to prepare plasmid DNA from 1 ml of culture. It is based on elements of the alkaline extraction procedure of Birnboim & Doly (1979), an old observation that linear DNA is efficiently precipitated in alkaline magnesium salts (Studier, 1965), and the well-known rapid lysis of cells by boiling in 1 o/0 (w/v) sodium dodecyl sulfate. The resulting plasmid DNA is suitable for transformation and restriction analysis, and has been used for sequencing. Samples are processed in 15 ml polypropylene tubes and centrifugation is at room temperature in an Eppendorf-type centrifuge. The 1 ml of culture, usually grown by standing overnight at 37°C in MSZB (Studier & Moffatt, 1986) plus appropriate antibiotic, is centrifuged for 30 s, the supernatant removed by aspiration, and the pellet suspended in 100 ~1 of water. To this suspension is added 100 ~1 of @l M-NaOH, 10 mM-EDTA, 2% sodium
dodecyl sulfate, which is mixed immediately by vortexing because the samples rapidly become viscous. The tube is heated for 2 min in a boiling water-bath, 50 ~1 of 1 M-MgCl, is mixed in by vortexing, and the tube is placed on ice for 2 min. The precipitate is pelleted bj centrifuging for 30 s, 50 ~1 of 5 M-potassium acetate is mixed into the supernatant in the same tube by brief vortexing, and the tube is again placed on ice for 2 min. After centrifuging another 30 s, the supernatant is removed to a new tube containing @6 ml of 95% (v/v) ethanol, mixed by vortexing and placed on ice for 2 min. The tube is centrifuged for 1 min, the supernatant is removed by aspiration, and @5 ml of 70°i, ethanol is added. vortexed, centrifuged for 1 min and removed by aspiration. Residual solvent is removed under vacuum for 5 to 10 min and the pellet dissolved in a convenient volume of the desired solvent. t)ypicallg 50 ~1 of 10 mw-Tris, @l mM-EDTA (pH %O). The basic procedure can take less than 30 min but samples can be left longer on ice at any stage, or even at room temperature instead of on ice, which can be (*OILvenient when preparing many samples. The resulting plasmid DNA should be essentially free of chromosomal DNA but will contain RNA fragments, which can be removed by treatment with RNase. (e) Plasmid
tracsformutiotw
Plasmids were introduced into cells bv treat,ment with calcium chloride as described by We&ink rt a,l. (I 974). When testing for ability to establish target plasmids. transformability of the cells was verified with a plasmid known to be tolerated, and transforming capacity of thr plasmid preparation was verified on isogenic cells that lacked the gene for T7 RNA polymerase. all in t,he samr experiment.
3. Results and Discussion (a) 7’7 lysozyme clo?Lrs Plasmids pLysS and pLysL provide a low level of T7 lysozyme to uninduced BLZl(DE3), and pLysE and pLysH provide a considerably higher level, as apparent in pulse-labeled proteins resolved by gel electrophoresis of total cell extracts (not shown). The difference in lysozyme level is consistent with a higher level of expression when lysozyme mRXA can be transcribed from the tet promoter than when the gene is in the opposite orientation (Fig. 1). The lower level of lysozyme provided by pLysS or pLysL has little effect on growth of the cell, but the higher level provided by pLysE or pLysH can reduce the growth rate or increase lag time. All of these lysozyme plasmids are easily maintained under selection by chloramphenicol; none of them causes apparent lysis of the culture during growth or at saturation nor interferes with transformation by compatible plasmids. Plasmids pLysS and pLysE have the @H promoter for T7 RNA polymerase immediately following the lysozyme gene, whereas pLysL and pLysH lack this promoter (or any promoter for T7 RNA polymerase: Fig. 1). Therefore. T7 RNA polymerase should be able to express some lysozyme mRNA from pLysS and pLysE by transcribing
T7 Lysozyme Improves a T7 Expression System
39
vectors (Rosenberg et al., 1987), so most transcription by T? RNA polymerase should be directed toward the target gene when pLysS or pLysE is in the same cell with target plasmids derived from the PET vectors. Plasmids pLysS and pLysE were made before pLysL and pLysH, and most of the work reported here was done with them. (b) T7 lysozyme increases tolerance for target genes
PWE
PLYSS
tet r-L
/s
TA
lysoryme
cat
cat \,
0 PLYSH
Pf-YSL
Figure 1. Plasmids
pLysS,
pLysE,
pLysL
and pLysH.
around the plasmid (Studier & Rosenberg, 1981; McAllister et al., 1981; Studier & Moffatt, 1986), but should not express any lysozyme mRNA from pLysL and pLysH. The 43.8 promoter is weak relative to the 420 promoter (McAllister et al., 1981), the promoter used in the PET expression
The target genes used to explore the effects of T7 lysozyme on the inducible T7 expression system were the genes of bacteriophage T7, some of which would be expected to be very toxic to the cell. Each of the target plasmids was tested for ability to be established and maintained in BL21, BL21(DE3), BL21(DE3)pLysS and BL21(DES)pLysE, and many were tested also in the equivalent strains based on HMS174. Of a set of 54 target plasmids, 13 were too toxic to be established in the expression host BL21(DE3), and several others were unstable or showed other signs of toxicity (Table 1). However, each target plasmid could be established in BL21 itself, consistent with the toxicity being due to target gene expression directed by basal T7 RNA polymerase in the expression host. In the presence of pLysS, the number of target plasmids that could not be established at all was reduced from 13 to 7, and others became more stable; in the presence of pLysE, additional target plasmids were stabilized and only four could not be established at
Table 1 Effect of T7 lysozyme on stability of cloned
T7 genes that are toxic or unstable in BL21 (D.E3) Plasmid maintenance
Promoters ahead of gene 410 410 410 410 410 410, $10, 410 410, 410 410 410, 410 410, $10 l$lO, ;:z 410 410 410 410
&3 Ql.6 $2.5 43.X 44.3 44.7
Functiont
Gene o-4 o-5 o-6 I.1 1.1, 1.2 1-3 1.6 2 2-5 2.8 3 3.8 4B 4-3 4.5 47 55.3 F7 59 17.5 18
Plasmid PAR number
DNA polymerase
2919 2478 2499 3491 2554 2046 3493 2048 3506 3480 2471 2562 3708 3471 2503 2606 2441 2298
Lysis DNA ends
2421 3420 2412 2652
Ligase RNA pol. inhib. SSB Endonucleaae Helicase
and cell growth3 BL21(DE3) PLYSS
BL21(DE3) PLYSE
BL21
BL21(DE3)
G?
LL Ksat? T Ksat Ksat T U -
U + + + + + Ksat? U + tiny ii -
LL + + + + + +
KU -
t Ksat? -
L:. + +
T? + T’ + + + + Ti K + + + li + K? A
G-? K + + + -+
t Dunn & Studier (1983), and unpublished work. SSB, single-stranded DNA-binding protein; pol. inhib., polvmerase inhibition. 1 Symbols for plasmid stabilitg and effect on growth of each host cell: -, plasmid cannot be established: U. piasmid will transform cells but is rerY unstable; T, plasmid is somewhat toxic but can be maintained; LL. cultures grow but lawns IYse or cells die upon plating: K. manY cells from growing cultures do not form colonies; Ksat. cultures grow but cells die at saturation: + plasmid is stable.
P. W. Studier
40
BL21(DE3)
BL21(DE3) PLYSL
BL21(DE3) PLYSS
BL21(DE3) PLYSH
BL21(DE3) PLYSE
IOB IOA
Figure 2. Basal and induced levels of gene 10 protein expressed from pAR3625 in BL21(DE3) itself or caontaining pLysL, pLysS, pLysH or pLysE. Plasmid pAR3625 contains T7 gene 10 under control of its natural 410 promoter and T4 transcription terminator. Cultures were grown at 37°C in MSZB containing 20 pg ampicillin/ml and 10 ng chloramphenicol/ml, where appropriate, to an A6s0 of about 66 to 0% and induced with 64 mnn-TPTG. Samples (20 ~1) of culture were added to 10 ~1 of 3 x concentrated sample buffer, heated for 2 min in a boiling water-bath, subjected to electrophoresis in the presence of sodium dodecyl sulfate in a polyacrylamide gel containing a loo& to 200/;, (W/V) gradient of acrylamide and a 5% (w/v) stacking gel, and stained with Coomassie brilliant blue esentially as described (Studier $ Moffatt, 1986). Each set of samples was collected immediately before and 05. 1, 2 and 3 h after induction.
all. The most toxic target genes were T7 genes 0.4, O-6 and 5.3, all of unknown function, and the gene I.1 and 1.2 combination, which may have some role in replication (Saito & Richardson, 1981; Huber et al., 1988). The stabilizing effect of pLysS and pLysE on toxic target plasmids is presumably because T7 lysozyme inhibits the small amount of T7 RNA polymerase present in the uninduced cell: which in turn reduces basal expression of the target gene. Comparisons of uninduced target protein levels from target plasmids that can be established in all three expression hosts show that pLysS and pLysE both cause a substantial reduction in basal expression of target genes (Fig. 2 and other target genes not shown; see also Dubendorff & Studier, 1991a,b, the accompanying papers). Most target plasmids seemed to behave similarly in the expression strains based on HMS174 or BL21. In some cases, however, a target gene was less toxic in the HMS174 derivatives. The most striking example was T7 gene 18, which specifies a protein involved in the maturation of T7 DNA (Studier, 1972). The gene 18 target plasmid is highly toxic to
BL21 (Table 1) but’ is easily established in HMSl74(DE3); gene 18 is so innocuous in HMS174 that a, clone of it expressed from the teb promoter of pRR322 is tolerated. The basis for such differences in toxicity is not known, but. a possible explanation in at least some cases has been suggested by Stewart Shuman (personal communication): a small amount of the target gene product might cause induction of the DE3 prophage and thereby kill the cell. Induction of lambda-type prophages requires wcA (Roberts & Devoret, 1983), which is functional in BL21 but not in HMS174. (c) induction of target prenence of T7
proteins
iv1 thr
lysozyme
The effect of each of the lysozyme plasmids on expression and accumulation of T7 gene 10 protein, the major T7 capsid protein. is shown in Figure 2. In the absence of any lysozyme plasmid, a prominent band of gene 10 protein is evident in t,hc sample taken immediately before induction) indeating substantial expression of target protein in the uninduced cell. This band is weaker but still signifi-
41
T7 Lysozyme Improves a T7 Expression System cant in the presence of pLysL and very weak but still detectable (in the original, if not in the photograph) in the presence of pLysS. Little if any gene 10 protein was apparent in the uninduced cells carrying pLysH or pLysE. Clearly, the presence of T7 lysozyme can reduce basal expression of target genes in BL21(DE3). Upon induction, the level of the gene 1OA protein and its frameshifted relative 10B (Dunn & Studier, 1983) increased substantially in all cases, but with notable differences. Although a large amount of gene 10 protein was accumulated within three hours after induction in the absence of any lysozyme plasmid, even more accumulated in the presence of pLysL, and more still in the presence of pLysS, pLysH or pLysE. These differences in total accumulation may reflect differences in the state of the cells at the time of induction, as indicated by colonyforming ability. Although all of the cultures were growing well, as indicated by rates of increase in turbidity, titers at equivalent optical density were very different: the culture having no lysozyme plasmid gave only about 7% as many colonies as the cultures where the cells carried pLysS or pLysE, or from a culture where the cells carried neither the gene 10 target plasmid nor a lysozyme plasmid; the culture with pLysL had about 30% the colonyforming efficiency and that with pLysH about 60%. The relatively high basal levels of gene 10 protein in cells without a lysozyme plasmid, or in those containing pLysL, were presumably responsible both for the reduced colony-forming ability and for the decreased ability to respond to induction. Although as much or more gene 10 protein accumulated in three hours in the presence of pLysH and pLysE as in the other cultures, the initial was delayed about increase after induction 30 minutes (Fig. 2). This delay presumably reflects the time needed to produce enough T7 RNA polymerase to overcome the inhibitory effect of the relatively high level of lysozyme in the cell at the time of induction.
The greater induction of gene 10 protein in the presence of pLysE than in the absence of lysozyme is unusual and presumably reflects differences in the health of the cells at the time of induction, as discussed above. Other target genes controlled by the same 410 promoter in the PET-1 vector typically accumulate considerably less target protein in the presence of pLysE than in the presence of pLysS, or in the absence of any lysozyme plasmid, even after longer induction. Apparently, the level of lysozyme provided by pLysE is high enough to inhibit a substantial fraction of the t’ranscription capacity of fully induced T7 RNA polymerase in BL21(DE3). Even so, target mRNAs that are translated efficiently, such as the gene 10 mRNA, can produce very high levels of protein in the presence of pLysE. A wide variety of target genes besides the T7 genes described here have been established and expressed to high levels in the PET expression vectors in BL21(DE3)pLysS. In most cases examined, the presence of pLysS has little effect on the amount of target protein accumulated, although full expression is often delayed a few minutes. Apparently, induction rapidly produces enough T7 RNA polymerase to overwhelm the inhibitory effect of the relatively low level of T7 lysozyme, and the $3.8 promoter in pLysS does not compete seriously against the 410 promoter in the PET vectors. (d) Modulation
of f&y
induced target
gene expression
T7 RNA polymerase in the absence of a target promoter is not particularly toxic to E. coli, but added target DNA can be transcribed so actively that the cell becomes unable to continue dividing even if the target RNA or protein is innocuous. Thus, the T7 expression hosts BL21 (DE3) or HMS174(DE3) are able to grow continuously and form colonies in the presence of inducer! but the same cells containing PET-1 cannot, (Table 2).
Table 2 Growth of cells containing different target plasmids, as a function RNA polymerase activity
of the level of T?
Growth of cells that contain plasmidj
Host cellt BL21 BLel(DE3)pLysE BLSl(DE3)pLysS BL21(DE3) BLSl(DE3)pLysE BLBl(DE3)pLysS BL21(DE3)
IPTG in plate
pBR322
0 0 0 0 + + +
+ + + + + + +
PET-3 WO-T4) + + + + + + 0
PET-1 (410)
54 clones of T7 genes§
+ + + + + 0 0
54 50 47 41 13 0 0
t Listed in order of increasing steady-state T7 RNA polymerase activity. $ Colony formation at the same efficiency in the presence or absence of ampicillin is taken to mean growth. $ The number of clones that allowed growth, from a set of 54 clones of T7 genes under control of a T7 promoter (usually 410).
F. W. Studier
42
TSP
pLysL TSP
pLysS TSP
pLysH TSP
pLysE TSP
Figure 3. Cell extracts from BL21(DE3) itself or containing pLysL, pLysS, pLysH or pLysE. Cultures were grown in MSZB containing 25 pg chloramphenicol/ml, where appropriate, to an A,,, of about 1. Samples (1 ml) of culture were centrifuged for @5 min in an Eppendorf centrifuge, the supernatant removed by aspiin 05 ml of and the cells suspended ration, 50 mm-Tris . HCl (pH %O), 2 mM-Na,EDTA. To the suspended cells was added 5 ~1 of 10% Triton X-100 to produce a concentration of 0.1%. The samples containing a lysozyme plasmid became very viscous within a few minutes but the sample without any lysozyme plasmid did not. After 15 min at room temperature (about 25”C), 5 ~1 of 1 M-MgSO, was added to each of the samples. The viscosity rapidly decreased, presumably due to digestion of the DNA by endogenous nucleases. A sample was saved for analysis of the total culture and the remainder was centrifuged for 1 min in an Eppendorf centrifuge, the
supernatant removed and the pellet resuspended in the same volume of 50 mm-Tris . HCl (pH 8.0). :! mMSa,EDTA. Samples of the total (T). supernatant’ (S) and resuspended pellet (P), each equivalent to about 20 ~1 of original culture, were analyzed by gel electrophoresis as described in the legend to Fig. 2.
Addition of pLysH or pLysE relieves the toxic effect of PET-1 but pLysL and pLysS do not. Apparently, the level of T7 lysozyme provided by pLysH or pLysE reduces the maximum transcription to a level where induced
cells can continue
to
grow, as long as the target gene products are not themselves toxic. Although pLysS does not permit the growth of induced cells that carry PET-1, it, does permit the growth of cells that carry PET-3 (Table 2), a target plasmid similar to PET-1 but which carries the T+
transcription terminator in addition to the $10 promoter (Rosenberg et al., 1987). Apparently. pLysS reduces the fully induced level of transcription sufficiently to allow growth if the target plasmid contains a terminator but not if it does not. Whatever the magnitude of this reduction, it appears to have little effect’ on the ultimate rate or total accumulation of most target proteins expressed from PET-I or PET-3. T7 RNA polymerase is capable of producing so much target, mRNA in the absence of lysozyme (Studier &, Moffatt, 1986) that perhaps the level of transcription can be reduced appreciably with little effect on the production of most target proteins. Plasmid pLysL is less effective than pLysS in allowing the growth of induced BL21(DE3) carrying PET-3: colonies eventually form, but much more slowly than when the cells contain pLysS. This observation, together with the higher basal level of gene 10 protein (Fig. 2), indicates that, pLysL is less effective than pLysS in reducing transcription from the target promoter in both the induced and uninduced cell. Some target plasmids can also be estahlished in BL21(DE3) in the presence of pLysS but not pLysL. The greater effectiveness of pLysS is most likely due to the presence of the &I.8 promoter, which could reduce expression from the target promoter in two ways: it could compet,e with the target promoter for transcription by T7 RNA polymerase, and such transcription could increase the level of T7 lysozyme by directing synthesis of lysozyme mRNA by T7 RNA polymerase transcribing around the plasmid (see Fig. i ). The ability of a sufficiently high level of T7 lysozyme to permit continuous growth of induced expression hosts that contain target plasmids adds another dimension to the T7 expression system. Individual target genes differ greatly in toxicity and in translational efficiency. At least 13 T7 genes are sufficiently innocuous that BL21(DE3) containing both the target plasmid and pLysE can continue to grow fully induced (although none of them could be tolerated in the presence of pLysS: Table 2). Thus. pLysE can allow some target proteins to be produced from continuous culture, if that would be advantageous. On the other hand, some target products kill the cell by interfering with specific cell functions, and the action of these gene products could be analyzed by inducing them in the presence of pLysE, without the complication of a lethal level of transcription by T7 RNA polymerase. (e) D$ferent steady-state of transcription
levels
The ability to modulate transcription activity of T7 RNA polymerase by supplying T7 lysozyme from a plasmid means that several different steadystate levels of transcription are accessible in a DE3 lysogen. Six such levels are represented in Table 2. where the differences are reflected in the numbers of different cloned T7 genes that can be maintained at each level of transcription. The lowest level is in
T7 Lgsozyme Improves a T7 Expression System uninduced BL21(DE3)pLysE, where 52 of 56 target plasmids could be maintained (including PET-1 and PET-S); the highest level is in induced BL21(DE3) itself, where none of the 56 target plasmids allows continued growth of the cell. The lowest level of transcription of target genes in PET-1 is not as low as in the complete absence of T7 RNA polymerase, since four of the target plasmids that could be established in BL21 could not be established in BL21(DES)pLysE. The highest level of transcription that allows stable maintenance of innocuous target genes in PET-1 is in induced BLBl(DES)pLysE, where 13 target plasmids could be maintained. This level of transcription appears to be somewhat higher than if the gene were cloned in the BamHI site of pBR322 under control of the tet promoter, where many T7 genes cannot be cloned (Studier t Rosenberg, 1981): ten of the genes that could be tolerated in induced BL21(DE3)pLysE have been cloned under control of the tet promoter, plus some that could not. (f) T7 lysozyme facilitates production of cell extracts T7 lysozyme cuts a bond in the cell wall of E. coli (Inouye et al., 1973) besides inhibiting T7 RNA polymerase. Nevertheless, cells are able to tolerate substantial levels of lysozyme, apparently because the enzyme is unable to penetrate the cell membrane to reach its substrate in the peptidoglycan layer. Treatments that disrupt membranes cause lysis of cells that contain even very small amounts of T7 lysozyme (Moffatt & Studier, 1987). The presence of any of the T7 lysozyme plasmids can facilitate the preparation of cell extracts for purification of target proteins. Simply collecting the cells by centrifugation and suspending them in 50 mM-Tris . HC1, 2 mM-Na,EDTA (pH SO) makes them highly susceptible to lysis by agents that disrupt cell membranes. Thorough lysates of such cell suspensions can be made by freezing and thawing or by adding agents such as @l y. (v/v) Triton X-100 (Fig. 3), @S% (w/v) deoxycholate or 1 y. (v/v) chloroform. This ease of preparing cell extracts can make it advantageous to carry pLysS or pLysL in the cell even when it is not required for stabilizing the target plasmid. I thank K. Takayama for constructing pLysS and pLysE, X. Zhang for constructing pLysL and pLysH, A. Rosenberg for constructing most of the T7 target plasmids, and J. Paparelli and K. Griffin for expert technical assistance. This work was supported by the Office of Health and Environmental Research of the United States Department of Energy.
References Birnboim, H. C. & Doly, J. (1979). A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucl. Acids Res. 7, 1513-1523. Chamberlin, M., McGrath, J. & Waskell, L. (1970). New
43
RNA polymerase from Escherichia coZi infected with bacteriophage T7. Nature (London), 228, 227-231. Chang, A. C. Y. & Cohen, S. N. (1978). Construction and characterization of amplifiable multicopy DNA cloning vehicles derived from the P15A cryptic miniplasmid. J. BacterioZ. 134, 1141-l 156.
Chen, W., Tabor, S. & Struhl, K. (1987). Distinguishing between mechanisms of eukaryotic transcriptional activation with bacteriophaage T7 RNA polymerase. Cell, 50, 1047-1055. Dubendorff, J. W. t Studier, F. W. (1991a). Controlling basal expression in an inducible T7 expression system by blocking the target T7 promoter with lac repressor. J. Mol. Biol. 219, 45-59. DubendorfT, J. W. & Studier, F. W. (199lb). Creation of a T7 autogene. Cloning and expression of the gene for bacteriophage T7 RNA polymerase under control of its cognate promoter. J. Mol. Biol. 219, 6lL68. Dunn, J. J. & Studier, F. W. (1983). Complete nucleotide sequence of bacteriophage T7 DNA and the locations of T7 genetic elements. J. Mol. Biol. 166, 477-535; and erratum (1984). J. Mol. BioZ. 175, 11 l-112. Dunn, J. J., Krippl, B., Bernstein, K. E., Westphal, H. & Studier, F. W. (1988). Targeting bacteriophage T7 RNA polymerase to the mammalian cell nucleus. Gene, 68, 259-266. Fuerst, T. R.. Niles, E. G., Studier, F. W. & Moss, B. (1986). Eukaryotic transient-expression system based on recombinant vaccinia virus that synthesizes bacteriophage T7 RNA polymerase. Proc. Nat. Acad. Sci., U.S.A. 83, 8122-8126. Huber, H. E., Beauchamp, B. B. & Richardson, C. C. (1988). Escherichiu coli dGTP triphosphohydrolase is inhibited by gene 1.2 protein of bacteriophage T7. J. Biol. Chem. 263, 13549-13556. Inouye, M., Arnheim, N. & Sternglanz, R. (1973). Bacteriophage T7 lysozyme is an N-acetylmuramyl-L-alanine amidase. J. Biol. Chem. 248, 7247-7252. McAllister, W. T., Morris, C., Rosenberg, A. H. & Studier, F. W. (1981). Utilization of bacteriophage T7 late promoters in recombinant plasmids during infection. J. Mol. Biot. 153, 527-544. Moffatt, B. A. & Studier, F. W. (1987). ‘T7 lysozyme inhibits transcription by T7 RNA polymerase. Cell, 49, 221-227. Roberts, J. W. & Devoret, R. (1983). Lysogenic induction. In Lambda II (Hendrix, R. W., Roberts, J. W., Stahl, F. W. & Weisberg, R. A., eds), pp. 123-144, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY Rose, R. E. (1988). The nucleotide sequence of pACYCl84. Nucl. Acids Res. 16, 355. Rosenberg, A. H., Lade, B. N., Chui, D.-s., Lin, S.-W., Dunn. J. J. & Studier, F. W. (1987). Vectors fat selective expression of cloned DNAs by T7 RNA polymerase. Gene, 56, 125-135. Saito, H. & Richardson, C. C. (1981). Genetic analysis of gene 1.2 of bacteriophage T7: Isolation of a mutant of Escherichia coli unable to support the growth of T7 gene 1.2 mutants. J. Viral. 37, 343-351. Studier, F. W. (1965). Sedimentation studies of the size and shape of DNA. J. Mol. Biol. 11, 373-390. Studier, F. W. (1972). Bacteriophage T7. Science, 176, 367-376. Studier, F. W. & Rosenberg, A. H. (1981). Genetic and physical mapping of the late region of bacteriophage T7 DNA by use of cloned fragments of T7 DNA. J. Mol. Biol. 153, 503-525.
44
F. W. Studier
Studier, F. W. & Moffatt, B. A. (1986). Use of bacteriophage T7 RNA polymerase to direct selective highlevel expression of cloned genes. J. Mol. Biol. 189. 113-130. Tabor, S. & Richardson, C. C. (1985). A bacteriophage TT Rh’A polymerase/promoter system for controlled
exclusive expression of specific genes. I’roc. Xal. Amd. Sci., U.S.A. 82, 1074-1078. Wensink, P. C., Finnegan. D. .J.. Donelson, J. E. & H ogness, D. S. (1974). A system for mapping DNA sequences in the chromosomes of’ Drosophiln melanognster.
Edited by R. Bchleif
(‘ell,
3, 31%325.
Use of Bacteriophage T7 Lysozyme to Improve an Inducible T7 Expression System F. William Studier Biology Department Brookhaven National Laboratory Upton, NY 11973, U.S.A. (Received 9 August
1990; accepted 11 January
1991)
Bacteriophage T7 lysozyme, a natural inhibitor of T7 RNA polymerase, can reduce basal activity from an inducible gene for T7 RNA polymerase and allow relatively toxic genes to be established in the same cell under control of a T7 promoter. Low levels of T7 lysozyme supplied by plasmids pLysS or pLysL, which are compatible with the PET vectors for expressing genes from a T7 promoter, are sufficient to stabilize many target plasmids and yet allow high levels of target protein to be produced upon induction of T7 RNA polymerase. Higher levels of lysozyme supplied by plasmids pLysE or pLysH reduce the fully induced activity of T7 RNA polymerase such that induced cells can continue to grow Different configurations of the and produce innocuous target proteins indefinitely. expression system can maintain several different steady-state levels of target gene expression. The presence of T7 lysozyme has the further advantage of facilitating the lysis of cells in preparing extracts for purification of target gene products. Keywords: T7 lysozyme; T7 RNA polymerase; T7 expression system; toxic target genes; cloned genes
1. Introduction
the range and versatility expression system.
Bacteriophage T7 RNA polymerase actively and selectively directs transcription from a relatively large promoter that is unlikely to be found in DNA unrelated to T7 (Chamberlin et al., 1970). This selectivity provides the basis for gene expression systems in Escherichia coli (Tabor & Richardson, 1985; Studier & Moffatt, 1986; Rosenberg et al., 1987) and potentially in a variety of cell types (Fuerst et al., 1986; Chen et al., 1987; Dunn et al., 1988). Configurations where T7 RNA polymerase is inducible are convenient for expressing a wide variety of target genes. However, the polymerase is so active that even a small basal level in the uninduced cell can prevent relatively toxic target genes from being established or make them difficult to maintain. Furthermore, the induced level of transcription of target DNA becomes so high that, even if the target gene is innocuous, the cell eventually stops growing and dividing. T7 lysozyme is a bifunctional protein that cuts a bond in the cell wall of E. coli (Inouye et al., 1973) and selectively inhibits T7 RNA polymerase by binding to it, a feed-back mechanism that ensures a controlled burst of transcription during T7 infection (Moffatt & Studier, 1987). These activities of T7 lysozyme are now shown to be useful for increasing
of
an
inducible
T7
2. Materials and Methods (a) The T7 expression
system
T7 expression hosts were E. coli BL21(DE3) and HMS174(DE3), which are lysogens of DE3, a phage lambda derivative that carries the gene for T7 RNA polymerase under control of the ZacUVS promoter (Studier & Moffatt, 1986). Target genes were cloned under control of the strong $10 promoter for T7 RNA polymerase in plasmid PET-1, a derivative of plasmid pBR322 that confers resistance to ampicillin (Rosenberg et al., 1987). The 410 promoter in PET-1 is directed counterclockwise across the BamHI site of pBR322, opposed to the tet promoter. Plasmid PET-3 is similar to PET-1 except that it also carries the T$ transcription terminator located just past the BumHI site (Rosenberg et aZ., 1987). Conditions for growing cultures, inducing synthesis of T7 RNA polymerase and expressing target genes have been described (Studier & Moffatt, 1986) and here in the Figure legends. Ability of induced cells to form colonies was tested by adding uninduced cells to 2.5 ml of melted top agar containing 1 mM-IPTGt and 9.5 mg of ampicillin
t Abbreviation used: IPTG, isopropyl-B-othiogalactopyranoside. 37
00X-2836/91
/090037-08
$03.00/O
0
1991 Academic
Press Limited
38
F. W. Studier
and plating onto 20 ml of solidified lacked these additives.
bottom
agar that
(b) Tr lysozyme clones T7 gene 35, the gene for T7 lysozyme, was cloned in the BarnHI site of pACYC184 (Chang & Cohen, 1978; Rose, 1988), a plasmid that is compatible with pBR322-based plaamids such as the PET plasmids. A fragment from plasmid pAR.410 (Studier & Rosenberg, 1981: base-pairs 10,665 to 11,296 of T7 DNA) contains the coding sequence for the last 13 amino acids of T7 gene 3, the entire coding sequence of gene 3.5 (base-pairs 10,706 to 11,161), the 43.8 promoter for T7 RNA polymerase, and the coding sequence for the first 24 amino acids of gene 3.8 (Dunn & Studier, 1983). Plasmid pLysE (Expressed) has this fragment oriented so that lysozyme mRNA is transcribed from the tet promoter of pACYCl84; pLysS (Silent) has the fragment in the opposite orientation (Fig. 1). A shorter gene 3.5 fragment, which ends at base-pair 11,164 and therefore lacks the 43.X promoter and the beginning of gene 3.8, was also cloned. Plasmid pLysH (High level) has this shorter fragment oriented so that lysozyme mRNA is transcribed from the tet promoter; pLysL (Low level) has the fragment in the opposite orientation. These lysozyme plasmids confer resistance to chloramphenicol; cells containing them are typically grown in medium containing 10 to 25 pg chloramphenicol/ml or are selected on plates to which 250 to 625 pg of chloramphenicol has been added to the top agar. (c) Target
genes
Each of the genes of bacteriophage T7 (Dunn $ Studier. 1983) has been cloned individually under control of a class III T7 promoter (Studier & Rosenberg, 1981; and work to be described elsewhere). Genes naturally adjacent to a class III promoter were cloned along with their promoter in the BamHI site of pBR322; the other genes were cloned under control of the 410 promoter in the BarnHI site of PET-1. Some genes cloned in PET-1 were not separated from an adjacent class IT promoter and were therefore under control of both 410 and the class II promoter. A set of 54 target pl&smids, representing 53 different T7 genes (all except the genes for T7 RNA polymerase, T7 lysozyme and the T7 protein kinase) was used in this work. including a clone of genes I.1 and 1.2 under control of $10. a combination more toxic than either gene cloned individually. (d) Isolation
of plasmid
DNA
The following rapid and convenient procedure was used to prepare plasmid DNA from 1 ml of culture. It is based on elements of the alkaline extraction procedure of Birnboim & Doly (1979), an old observation that linear DNA is efficiently precipitated in alkaline magnesium salts (Studier, 1965), and the well-known rapid lysis of cells by boiling in 1 o/0 (w/v) sodium dodecyl sulfate. The resulting plasmid DNA is suitable for transformation and restriction analysis, and has been used for sequencing. Samples are processed in 15 ml polypropylene tubes and centrifugation is at room temperature in an Eppendorf-type centrifuge. The 1 ml of culture, usually grown by standing overnight at 37°C in MSZB (Studier & Moffatt, 1986) plus appropriate antibiotic, is centrifuged for 30 s, the supernatant removed by aspiration, and the pellet suspended in 100 ~1 of water. To this suspension is added 100 ~1 of @l M-NaOH, 10 mM-EDTA, 2% sodium
dodecyl sulfate, which is mixed immediately by vortexing because the samples rapidly become viscous. The tube is heated for 2 min in a boiling water-bath, 50 ~1 of 1 M-MgCl, is mixed in by vortexing, and the tube is placed on ice for 2 min. The precipitate is pelleted bj centrifuging for 30 s, 50 ~1 of 5 M-potassium acetate is mixed into the supernatant in the same tube by brief vortexing, and the tube is again placed on ice for 2 min. After centrifuging another 30 s, the supernatant is removed to a new tube containing @6 ml of 95% (v/v) ethanol, mixed by vortexing and placed on ice for 2 min. The tube is centrifuged for 1 min, the supernatant is removed by aspiration, and @5 ml of 70°i, ethanol is added. vortexed, centrifuged for 1 min and removed by aspiration. Residual solvent is removed under vacuum for 5 to 10 min and the pellet dissolved in a convenient volume of the desired solvent. t)ypicallg 50 ~1 of 10 mw-Tris, @l mM-EDTA (pH %O). The basic procedure can take less than 30 min but samples can be left longer on ice at any stage, or even at room temperature instead of on ice, which can be (*OILvenient when preparing many samples. The resulting plasmid DNA should be essentially free of chromosomal DNA but will contain RNA fragments, which can be removed by treatment with RNase. (e) Plasmid
tracsformutiotw
Plasmids were introduced into cells bv treat,ment with calcium chloride as described by We&ink rt a,l. (I 974). When testing for ability to establish target plasmids. transformability of the cells was verified with a plasmid known to be tolerated, and transforming capacity of thr plasmid preparation was verified on isogenic cells that lacked the gene for T7 RNA polymerase. all in t,he samr experiment.
3. Results and Discussion (a) 7’7 lysozyme clo?Lrs Plasmids pLysS and pLysL provide a low level of T7 lysozyme to uninduced BLZl(DE3), and pLysE and pLysH provide a considerably higher level, as apparent in pulse-labeled proteins resolved by gel electrophoresis of total cell extracts (not shown). The difference in lysozyme level is consistent with a higher level of expression when lysozyme mRXA can be transcribed from the tet promoter than when the gene is in the opposite orientation (Fig. 1). The lower level of lysozyme provided by pLysS or pLysL has little effect on growth of the cell, but the higher level provided by pLysE or pLysH can reduce the growth rate or increase lag time. All of these lysozyme plasmids are easily maintained under selection by chloramphenicol; none of them causes apparent lysis of the culture during growth or at saturation nor interferes with transformation by compatible plasmids. Plasmids pLysS and pLysE have the @H promoter for T7 RNA polymerase immediately following the lysozyme gene, whereas pLysL and pLysH lack this promoter (or any promoter for T7 RNA polymerase: Fig. 1). Therefore. T7 RNA polymerase should be able to express some lysozyme mRNA from pLysS and pLysE by transcribing
T7 Lysozyme Improves a T7 Expression System
39
vectors (Rosenberg et al., 1987), so most transcription by T? RNA polymerase should be directed toward the target gene when pLysS or pLysE is in the same cell with target plasmids derived from the PET vectors. Plasmids pLysS and pLysE were made before pLysL and pLysH, and most of the work reported here was done with them. (b) T7 lysozyme increases tolerance for target genes
PWE
PLYSS
tet r-L
/s
TA
lysoryme
cat
cat \,
0 PLYSH
Pf-YSL
Figure 1. Plasmids
pLysS,
pLysE,
pLysL
and pLysH.
around the plasmid (Studier & Rosenberg, 1981; McAllister et al., 1981; Studier & Moffatt, 1986), but should not express any lysozyme mRNA from pLysL and pLysH. The 43.8 promoter is weak relative to the 420 promoter (McAllister et al., 1981), the promoter used in the PET expression
The target genes used to explore the effects of T7 lysozyme on the inducible T7 expression system were the genes of bacteriophage T7, some of which would be expected to be very toxic to the cell. Each of the target plasmids was tested for ability to be established and maintained in BL21, BL21(DE3), BL21(DE3)pLysS and BL21(DES)pLysE, and many were tested also in the equivalent strains based on HMS174. Of a set of 54 target plasmids, 13 were too toxic to be established in the expression host BL21(DE3), and several others were unstable or showed other signs of toxicity (Table 1). However, each target plasmid could be established in BL21 itself, consistent with the toxicity being due to target gene expression directed by basal T7 RNA polymerase in the expression host. In the presence of pLysS, the number of target plasmids that could not be established at all was reduced from 13 to 7, and others became more stable; in the presence of pLysE, additional target plasmids were stabilized and only four could not be established at
Table 1 Effect of T7 lysozyme on stability of cloned
T7 genes that are toxic or unstable in BL21 (D.E3) Plasmid maintenance
Promoters ahead of gene 410 410 410 410 410 410, $10, 410 410, 410 410 410, 410 410, $10 l$lO, ;:z 410 410 410 410
&3 Ql.6 $2.5 43.X 44.3 44.7
Functiont
Gene o-4 o-5 o-6 I.1 1.1, 1.2 1-3 1.6 2 2-5 2.8 3 3.8 4B 4-3 4.5 47 55.3 F7 59 17.5 18
Plasmid PAR number
DNA polymerase
2919 2478 2499 3491 2554 2046 3493 2048 3506 3480 2471 2562 3708 3471 2503 2606 2441 2298
Lysis DNA ends
2421 3420 2412 2652
Ligase RNA pol. inhib. SSB Endonucleaae Helicase
and cell growth3 BL21(DE3) PLYSS
BL21(DE3) PLYSE
BL21
BL21(DE3)
G?
LL Ksat? T Ksat Ksat T U -
U + + + + + Ksat? U + tiny ii -
LL + + + + + +
KU -
t Ksat? -
L:. + +
T? + T’ + + + + Ti K + + + li + K? A
G-? K + + + -+
t Dunn & Studier (1983), and unpublished work. SSB, single-stranded DNA-binding protein; pol. inhib., polvmerase inhibition. 1 Symbols for plasmid stabilitg and effect on growth of each host cell: -, plasmid cannot be established: U. piasmid will transform cells but is rerY unstable; T, plasmid is somewhat toxic but can be maintained; LL. cultures grow but lawns IYse or cells die upon plating: K. manY cells from growing cultures do not form colonies; Ksat. cultures grow but cells die at saturation: + plasmid is stable.
P. W. Studier
40
BL21(DE3)
BL21(DE3) PLYSL
BL21(DE3) PLYSS
BL21(DE3) PLYSH
BL21(DE3) PLYSE
IOB IOA
Figure 2. Basal and induced levels of gene 10 protein expressed from pAR3625 in BL21(DE3) itself or caontaining pLysL, pLysS, pLysH or pLysE. Plasmid pAR3625 contains T7 gene 10 under control of its natural 410 promoter and T4 transcription terminator. Cultures were grown at 37°C in MSZB containing 20 pg ampicillin/ml and 10 ng chloramphenicol/ml, where appropriate, to an A6s0 of about 66 to 0% and induced with 64 mnn-TPTG. Samples (20 ~1) of culture were added to 10 ~1 of 3 x concentrated sample buffer, heated for 2 min in a boiling water-bath, subjected to electrophoresis in the presence of sodium dodecyl sulfate in a polyacrylamide gel containing a loo& to 200/;, (W/V) gradient of acrylamide and a 5% (w/v) stacking gel, and stained with Coomassie brilliant blue esentially as described (Studier $ Moffatt, 1986). Each set of samples was collected immediately before and 05. 1, 2 and 3 h after induction.
all. The most toxic target genes were T7 genes 0.4, O-6 and 5.3, all of unknown function, and the gene I.1 and 1.2 combination, which may have some role in replication (Saito & Richardson, 1981; Huber et al., 1988). The stabilizing effect of pLysS and pLysE on toxic target plasmids is presumably because T7 lysozyme inhibits the small amount of T7 RNA polymerase present in the uninduced cell: which in turn reduces basal expression of the target gene. Comparisons of uninduced target protein levels from target plasmids that can be established in all three expression hosts show that pLysS and pLysE both cause a substantial reduction in basal expression of target genes (Fig. 2 and other target genes not shown; see also Dubendorff & Studier, 1991a,b, the accompanying papers). Most target plasmids seemed to behave similarly in the expression strains based on HMS174 or BL21. In some cases, however, a target gene was less toxic in the HMS174 derivatives. The most striking example was T7 gene 18, which specifies a protein involved in the maturation of T7 DNA (Studier, 1972). The gene 18 target plasmid is highly toxic to
BL21 (Table 1) but’ is easily established in HMSl74(DE3); gene 18 is so innocuous in HMS174 that a, clone of it expressed from the teb promoter of pRR322 is tolerated. The basis for such differences in toxicity is not known, but. a possible explanation in at least some cases has been suggested by Stewart Shuman (personal communication): a small amount of the target gene product might cause induction of the DE3 prophage and thereby kill the cell. Induction of lambda-type prophages requires wcA (Roberts & Devoret, 1983), which is functional in BL21 but not in HMS174. (c) induction of target prenence of T7
proteins
iv1 thr
lysozyme
The effect of each of the lysozyme plasmids on expression and accumulation of T7 gene 10 protein, the major T7 capsid protein. is shown in Figure 2. In the absence of any lysozyme plasmid, a prominent band of gene 10 protein is evident in t,hc sample taken immediately before induction) indeating substantial expression of target protein in the uninduced cell. This band is weaker but still signifi-
41
T7 Lysozyme Improves a T7 Expression System cant in the presence of pLysL and very weak but still detectable (in the original, if not in the photograph) in the presence of pLysS. Little if any gene 10 protein was apparent in the uninduced cells carrying pLysH or pLysE. Clearly, the presence of T7 lysozyme can reduce basal expression of target genes in BL21(DE3). Upon induction, the level of the gene 1OA protein and its frameshifted relative 10B (Dunn & Studier, 1983) increased substantially in all cases, but with notable differences. Although a large amount of gene 10 protein was accumulated within three hours after induction in the absence of any lysozyme plasmid, even more accumulated in the presence of pLysL, and more still in the presence of pLysS, pLysH or pLysE. These differences in total accumulation may reflect differences in the state of the cells at the time of induction, as indicated by colonyforming ability. Although all of the cultures were growing well, as indicated by rates of increase in turbidity, titers at equivalent optical density were very different: the culture having no lysozyme plasmid gave only about 7% as many colonies as the cultures where the cells carried pLysS or pLysE, or from a culture where the cells carried neither the gene 10 target plasmid nor a lysozyme plasmid; the culture with pLysL had about 30% the colonyforming efficiency and that with pLysH about 60%. The relatively high basal levels of gene 10 protein in cells without a lysozyme plasmid, or in those containing pLysL, were presumably responsible both for the reduced colony-forming ability and for the decreased ability to respond to induction. Although as much or more gene 10 protein accumulated in three hours in the presence of pLysH and pLysE as in the other cultures, the initial was delayed about increase after induction 30 minutes (Fig. 2). This delay presumably reflects the time needed to produce enough T7 RNA polymerase to overcome the inhibitory effect of the relatively high level of lysozyme in the cell at the time of induction.
The greater induction of gene 10 protein in the presence of pLysE than in the absence of lysozyme is unusual and presumably reflects differences in the health of the cells at the time of induction, as discussed above. Other target genes controlled by the same 410 promoter in the PET-1 vector typically accumulate considerably less target protein in the presence of pLysE than in the presence of pLysS, or in the absence of any lysozyme plasmid, even after longer induction. Apparently, the level of lysozyme provided by pLysE is high enough to inhibit a substantial fraction of the t’ranscription capacity of fully induced T7 RNA polymerase in BL21(DE3). Even so, target mRNAs that are translated efficiently, such as the gene 10 mRNA, can produce very high levels of protein in the presence of pLysE. A wide variety of target genes besides the T7 genes described here have been established and expressed to high levels in the PET expression vectors in BL21(DE3)pLysS. In most cases examined, the presence of pLysS has little effect on the amount of target protein accumulated, although full expression is often delayed a few minutes. Apparently, induction rapidly produces enough T7 RNA polymerase to overwhelm the inhibitory effect of the relatively low level of T7 lysozyme, and the $3.8 promoter in pLysS does not compete seriously against the 410 promoter in the PET vectors. (d) Modulation
of f&y
induced target
gene expression
T7 RNA polymerase in the absence of a target promoter is not particularly toxic to E. coli, but added target DNA can be transcribed so actively that the cell becomes unable to continue dividing even if the target RNA or protein is innocuous. Thus, the T7 expression hosts BL21 (DE3) or HMS174(DE3) are able to grow continuously and form colonies in the presence of inducer! but the same cells containing PET-1 cannot, (Table 2).
Table 2 Growth of cells containing different target plasmids, as a function RNA polymerase activity
of the level of T?
Growth of cells that contain plasmidj
Host cellt BL21 BLel(DE3)pLysE BLSl(DE3)pLysS BL21(DE3) BLSl(DE3)pLysE BLBl(DE3)pLysS BL21(DE3)
IPTG in plate
pBR322
0 0 0 0 + + +
+ + + + + + +
PET-3 WO-T4) + + + + + + 0
PET-1 (410)
54 clones of T7 genes§
+ + + + + 0 0
54 50 47 41 13 0 0
t Listed in order of increasing steady-state T7 RNA polymerase activity. $ Colony formation at the same efficiency in the presence or absence of ampicillin is taken to mean growth. $ The number of clones that allowed growth, from a set of 54 clones of T7 genes under control of a T7 promoter (usually 410).
F. W. Studier
42
TSP
pLysL TSP
pLysS TSP
pLysH TSP
pLysE TSP
Figure 3. Cell extracts from BL21(DE3) itself or containing pLysL, pLysS, pLysH or pLysE. Cultures were grown in MSZB containing 25 pg chloramphenicol/ml, where appropriate, to an A,,, of about 1. Samples (1 ml) of culture were centrifuged for @5 min in an Eppendorf centrifuge, the supernatant removed by aspiin 05 ml of and the cells suspended ration, 50 mm-Tris . HCl (pH %O), 2 mM-Na,EDTA. To the suspended cells was added 5 ~1 of 10% Triton X-100 to produce a concentration of 0.1%. The samples containing a lysozyme plasmid became very viscous within a few minutes but the sample without any lysozyme plasmid did not. After 15 min at room temperature (about 25”C), 5 ~1 of 1 M-MgSO, was added to each of the samples. The viscosity rapidly decreased, presumably due to digestion of the DNA by endogenous nucleases. A sample was saved for analysis of the total culture and the remainder was centrifuged for 1 min in an Eppendorf centrifuge, the
supernatant removed and the pellet resuspended in the same volume of 50 mm-Tris . HCl (pH 8.0). :! mMSa,EDTA. Samples of the total (T). supernatant’ (S) and resuspended pellet (P), each equivalent to about 20 ~1 of original culture, were analyzed by gel electrophoresis as described in the legend to Fig. 2.
Addition of pLysH or pLysE relieves the toxic effect of PET-1 but pLysL and pLysS do not. Apparently, the level of T7 lysozyme provided by pLysH or pLysE reduces the maximum transcription to a level where induced
cells can continue
to
grow, as long as the target gene products are not themselves toxic. Although pLysS does not permit the growth of induced cells that carry PET-1, it, does permit the growth of cells that carry PET-3 (Table 2), a target plasmid similar to PET-1 but which carries the T+
transcription terminator in addition to the $10 promoter (Rosenberg et al., 1987). Apparently. pLysS reduces the fully induced level of transcription sufficiently to allow growth if the target plasmid contains a terminator but not if it does not. Whatever the magnitude of this reduction, it appears to have little effect’ on the ultimate rate or total accumulation of most target proteins expressed from PET-I or PET-3. T7 RNA polymerase is capable of producing so much target, mRNA in the absence of lysozyme (Studier &, Moffatt, 1986) that perhaps the level of transcription can be reduced appreciably with little effect on the production of most target proteins. Plasmid pLysL is less effective than pLysS in allowing the growth of induced BL21(DE3) carrying PET-3: colonies eventually form, but much more slowly than when the cells contain pLysS. This observation, together with the higher basal level of gene 10 protein (Fig. 2), indicates that, pLysL is less effective than pLysS in reducing transcription from the target promoter in both the induced and uninduced cell. Some target plasmids can also be estahlished in BL21(DE3) in the presence of pLysS but not pLysL. The greater effectiveness of pLysS is most likely due to the presence of the &I.8 promoter, which could reduce expression from the target promoter in two ways: it could compet,e with the target promoter for transcription by T7 RNA polymerase, and such transcription could increase the level of T7 lysozyme by directing synthesis of lysozyme mRNA by T7 RNA polymerase transcribing around the plasmid (see Fig. i ). The ability of a sufficiently high level of T7 lysozyme to permit continuous growth of induced expression hosts that contain target plasmids adds another dimension to the T7 expression system. Individual target genes differ greatly in toxicity and in translational efficiency. At least 13 T7 genes are sufficiently innocuous that BL21(DE3) containing both the target plasmid and pLysE can continue to grow fully induced (although none of them could be tolerated in the presence of pLysS: Table 2). Thus. pLysE can allow some target proteins to be produced from continuous culture, if that would be advantageous. On the other hand, some target products kill the cell by interfering with specific cell functions, and the action of these gene products could be analyzed by inducing them in the presence of pLysE, without the complication of a lethal level of transcription by T7 RNA polymerase. (e) D$ferent steady-state of transcription
levels
The ability to modulate transcription activity of T7 RNA polymerase by supplying T7 lysozyme from a plasmid means that several different steadystate levels of transcription are accessible in a DE3 lysogen. Six such levels are represented in Table 2. where the differences are reflected in the numbers of different cloned T7 genes that can be maintained at each level of transcription. The lowest level is in
T7 Lgsozyme Improves a T7 Expression System uninduced BL21(DE3)pLysE, where 52 of 56 target plasmids could be maintained (including PET-1 and PET-S); the highest level is in induced BL21(DE3) itself, where none of the 56 target plasmids allows continued growth of the cell. The lowest level of transcription of target genes in PET-1 is not as low as in the complete absence of T7 RNA polymerase, since four of the target plasmids that could be established in BL21 could not be established in BL21(DES)pLysE. The highest level of transcription that allows stable maintenance of innocuous target genes in PET-1 is in induced BLBl(DES)pLysE, where 13 target plasmids could be maintained. This level of transcription appears to be somewhat higher than if the gene were cloned in the BamHI site of pBR322 under control of the tet promoter, where many T7 genes cannot be cloned (Studier t Rosenberg, 1981): ten of the genes that could be tolerated in induced BL21(DE3)pLysE have been cloned under control of the tet promoter, plus some that could not. (f) T7 lysozyme facilitates production of cell extracts T7 lysozyme cuts a bond in the cell wall of E. coli (Inouye et al., 1973) besides inhibiting T7 RNA polymerase. Nevertheless, cells are able to tolerate substantial levels of lysozyme, apparently because the enzyme is unable to penetrate the cell membrane to reach its substrate in the peptidoglycan layer. Treatments that disrupt membranes cause lysis of cells that contain even very small amounts of T7 lysozyme (Moffatt & Studier, 1987). The presence of any of the T7 lysozyme plasmids can facilitate the preparation of cell extracts for purification of target proteins. Simply collecting the cells by centrifugation and suspending them in 50 mM-Tris . HC1, 2 mM-Na,EDTA (pH SO) makes them highly susceptible to lysis by agents that disrupt cell membranes. Thorough lysates of such cell suspensions can be made by freezing and thawing or by adding agents such as @l y. (v/v) Triton X-100 (Fig. 3), @S% (w/v) deoxycholate or 1 y. (v/v) chloroform. This ease of preparing cell extracts can make it advantageous to carry pLysS or pLysL in the cell even when it is not required for stabilizing the target plasmid. I thank K. Takayama for constructing pLysS and pLysE, X. Zhang for constructing pLysL and pLysH, A. Rosenberg for constructing most of the T7 target plasmids, and J. Paparelli and K. Griffin for expert technical assistance. This work was supported by the Office of Health and Environmental Research of the United States Department of Energy.
References Birnboim, H. C. & Doly, J. (1979). A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucl. Acids Res. 7, 1513-1523. Chamberlin, M., McGrath, J. & Waskell, L. (1970). New
43
RNA polymerase from Escherichia coZi infected with bacteriophage T7. Nature (London), 228, 227-231. Chang, A. C. Y. & Cohen, S. N. (1978). Construction and characterization of amplifiable multicopy DNA cloning vehicles derived from the P15A cryptic miniplasmid. J. BacterioZ. 134, 1141-l 156.
Chen, W., Tabor, S. & Struhl, K. (1987). Distinguishing between mechanisms of eukaryotic transcriptional activation with bacteriophaage T7 RNA polymerase. Cell, 50, 1047-1055. Dubendorff, J. W. t Studier, F. W. (1991a). Controlling basal expression in an inducible T7 expression system by blocking the target T7 promoter with lac repressor. J. Mol. Biol. 219, 45-59. DubendorfT, J. W. & Studier, F. W. (199lb). Creation of a T7 autogene. Cloning and expression of the gene for bacteriophage T7 RNA polymerase under control of its cognate promoter. J. Mol. Biol. 219, 6lL68. Dunn, J. J. & Studier, F. W. (1983). Complete nucleotide sequence of bacteriophage T7 DNA and the locations of T7 genetic elements. J. Mol. Biol. 166, 477-535; and erratum (1984). J. Mol. BioZ. 175, 11 l-112. Dunn, J. J., Krippl, B., Bernstein, K. E., Westphal, H. & Studier, F. W. (1988). Targeting bacteriophage T7 RNA polymerase to the mammalian cell nucleus. Gene, 68, 259-266. Fuerst, T. R.. Niles, E. G., Studier, F. W. & Moss, B. (1986). Eukaryotic transient-expression system based on recombinant vaccinia virus that synthesizes bacteriophage T7 RNA polymerase. Proc. Nat. Acad. Sci., U.S.A. 83, 8122-8126. Huber, H. E., Beauchamp, B. B. & Richardson, C. C. (1988). Escherichiu coli dGTP triphosphohydrolase is inhibited by gene 1.2 protein of bacteriophage T7. J. Biol. Chem. 263, 13549-13556. Inouye, M., Arnheim, N. & Sternglanz, R. (1973). Bacteriophage T7 lysozyme is an N-acetylmuramyl-L-alanine amidase. J. Biol. Chem. 248, 7247-7252. McAllister, W. T., Morris, C., Rosenberg, A. H. & Studier, F. W. (1981). Utilization of bacteriophage T7 late promoters in recombinant plasmids during infection. J. Mol. Biot. 153, 527-544. Moffatt, B. A. & Studier, F. W. (1987). ‘T7 lysozyme inhibits transcription by T7 RNA polymerase. Cell, 49, 221-227. Roberts, J. W. & Devoret, R. (1983). Lysogenic induction. In Lambda II (Hendrix, R. W., Roberts, J. W., Stahl, F. W. & Weisberg, R. A., eds), pp. 123-144, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY Rose, R. E. (1988). The nucleotide sequence of pACYCl84. Nucl. Acids Res. 16, 355. Rosenberg, A. H., Lade, B. N., Chui, D.-s., Lin, S.-W., Dunn. J. J. & Studier, F. W. (1987). Vectors fat selective expression of cloned DNAs by T7 RNA polymerase. Gene, 56, 125-135. Saito, H. & Richardson, C. C. (1981). Genetic analysis of gene 1.2 of bacteriophage T7: Isolation of a mutant of Escherichia coli unable to support the growth of T7 gene 1.2 mutants. J. Viral. 37, 343-351. Studier, F. W. (1965). Sedimentation studies of the size and shape of DNA. J. Mol. Biol. 11, 373-390. Studier, F. W. (1972). Bacteriophage T7. Science, 176, 367-376. Studier, F. W. & Rosenberg, A. H. (1981). Genetic and physical mapping of the late region of bacteriophage T7 DNA by use of cloned fragments of T7 DNA. J. Mol. Biol. 153, 503-525.
44
F. W. Studier
Studier, F. W. & Moffatt, B. A. (1986). Use of bacteriophage T7 RNA polymerase to direct selective highlevel expression of cloned genes. J. Mol. Biol. 189. 113-130. Tabor, S. & Richardson, C. C. (1985). A bacteriophage TT Rh’A polymerase/promoter system for controlled
exclusive expression of specific genes. I’roc. Xal. Amd. Sci., U.S.A. 82, 1074-1078. Wensink, P. C., Finnegan. D. .J.. Donelson, J. E. & H ogness, D. S. (1974). A system for mapping DNA sequences in the chromosomes of’ Drosophiln melanognster.
Edited by R. Bchleif
(‘ell,
3, 31%325.
