Gene, 106 (1991) l-6 (C; 1991 Elsevier

GENE

Science

Publishers

B.V. All rights reserved.

0378-l 119/91/$03.50

05054

Phage T4 expression vector: protection from proteolysis (T7 promoter;

cloning

vehicle;

recombinant

DNA;

toxicity of overproduced

products;

low basal expression)

Britta Swebilius Singer and Larry Gold Department

ofMolecular, Cellular, and Developnzental Biology, Universityof Colorado, Boulder, CO 80309 (U.S.A.)

Received by G. Gussin: 18 December Revised: 31 March 1991 Accepted: 15 April 1991

1991

SUMMARY

We have developed an efficient method for the expression of heterologous genes during infection by T4, a bacteriophage known to inhibit the proteolytic systems of Escherichiu cofi. This system enables us to clone genes in a plasmid expression vector and move them readily into T4. We have used IacZ as a reporter gene to show that both plasmid and phage exhibit low basal expression or high-level expression under the control of a T7 promoter. This system promises a possible solution to the problem of degradation and/or toxicity of overproduced proteins.

INTRODUCTION

A significant problem for the modern biologist arises when, after expending copious resources to clone a gene that encodes an important protein in an expression vector, it turns out that the protein is rapidly degraded by E. coli. The development of T4 as a cloning vehicle promises to circumvent this difficulty because T4 is one of the phages known to inhibit proteolysis of aberrant proteins during infection (Simon et al., 1978). Shub and his colleagues have used T4 as a cloning vector and observed stabilization of a BGal variant that is unstable in E. coli (Shub and Casna, 1985; Zeeh,

1989). We have developed

Correspondenceto: Dr. B.S. Singer, Department and

Developmental

Biology,

University

a T4 expression of Molecular,

of Colorado,

Cellular

Boulder,

CO

80309-0347 (U.S.A.) Tel. (303)492-4399; Fax (303)492-7744. Abbreviations:

acK, acriflavine

resistance;

BGal, p-galactosidase;

bp,

base pair(s); Cm, chloramphenicol; gp, gene product; HMC, methyl cytosine, IPTG, isopropyl-p-o-thiogalactopyranoside;

hydroxykb, kilo-

base(s) or 1000 bp; MCS, multiple cloning site; moi, multiplicity

ofinfec-

tion; nt, nucleotide(s); polyacrylamide-gel rifampicin; RNAP,

ORF, open reading

frame; P, promoter;

electrophoresis; RBS, ribosome-binding RNA polymerase; SDS, sodium dodecyl

terminator; wt, wild type; insertions into phage.

[ 1.denotes plasmid-carrier

PAGE, site; Rif, sulfate; r,

state; ( ), denotes

system that provides a number of advantages over the original methodology developed by Shub and Casna. Most important, both plasmid and phage produce very low basal levels and very high induced levels of expression. The plasmids that we constructed have a number of features that make them useful as cloning vehicles independent of their use in the T4 expression system. (1) They contain the MCS of pUC18/19. (2) They contain the T7 $10 promoter for very high level transcription under the control of the T7 gene 1 RNAP (Studier et al., 1990). They also have the T7 transcription terminator (Studier et al., 1990) which we will show is important for highest level expression. (3) They exhibit very low basal levels of transcription through the cloning region. (4) Because they are derived from pACYC184 (Chang and Cohen. 1978) they encode resistance to Cm and are very stable. Recombination between phage and plasmid is a reliable technique for replacing DNA resident in phage with homologous plasmid sequences (Mattson et al., 1977). We have flanked the cloning region with the C-terminal portion of the T4 rZZAgene and the N-terminal portion of the rIZB gene. When we select for phage recombinants that have acquired rll sequences (and the rI1 + phenotype) from the plasmid, the cloned sequence is moved into the phage chromosome as an unselected marker.

RESULTS

AND DISCUSSION

(a) The structure of the pDIP plasmids In this paper, we discuss a series of eight plasmids. pDIP18A-D are identical to pDIP19A-D except that pDIP18 has the MCS of pUC18 while pDIP19 has the MCS of pUC19, i.e., with the same restriction sites but in reverse orientation. The components of pDIP A-D are listed in Table I. Fig. 1 shows a restriction map of pDIP18/19D, the most ‘advanced’ plasmid in this series.

pDIP18/19D -4350 bp

(h) The pDIP plasmids as expression vectors High level production of /IGal in the pDIP : 1uc.Z plasmids requires transcription from the T7 promoter. Table II shows the levels of PGal activity that are achieved when the cells produce T7 RNAP. Cells were also harvested for SDS PAGE (see Fig. 2). With both assays, the highest level of /?Gal is produced by pDIP 19D : lucZ, the plasmid that has the T7 transcription terminator following the MCS.

Fig. 1. Restriction

map of pDIPl S/ 19D. Drawn

this map shows the location The MCS from pUC18/19

(c) The rpoC transcription terminator and basal levels of expression It is often as important to have very low levels of expression in the uninduced condition as it is to have very high levels of expression in the induced situation. Although the T7 promoter should be completely silent in the absence of T7 RNAP, genes cloned downstream of this promoter still exhibit basal expression because of readthrough transcription from plasmid promoters. In order to reduce readthrough transcription, we cloned the rpoC transcription terminator between ‘rlL4 and the origin of replication. In accordance with published reports (Albrechtsen et al.,

of the T7 promoter. from

Ml3

(Albrechtson

approximately

components

to scale,

of pDIP18/19D.

is shown at the top of the map, downstream

The rpoCt transcription

mpl8+rpoCt,

was

terminator

kindly

provided

et al., 1990). The rII gene fragments

to 415 and nt 439 to 873 (Pribnow

by

(T,,,,,.),

excised

C.L.

Squires

correspond

et al., 1981). ‘rIIA indicates

to nt 310 5’ trun-

cation of the rlIA gene and rIIL?’ indicates

3’ truncation

We used synthetic

for the final two amino

codons

oligodeoxynucleotides

and the stop codon of rIIA, retaining

The T7 $10 promoter transcription

(Pr,)

terminator

mot ORFD promoter

is derived

(Tr,)

(P,,,,)

of the rllB gene. acid

the wt amino acid sequence.

from pT7-1 (USBC).

is from PET-3 (Studier

The T7

et al., 1990). The

(Guild et al., 1988) and the RBS and first six

codons of rIIB’ are derived from synthetic oligodeoxynucleotides. With the exception of those in the MCS, all unique restriction sites are indicated. searched

We assembled with EuGene

found in the polylinker. with the exception

the presumptive (version

sequence

of the plasmid

3.2) for all of the 6-bp restriction

and sites

All of these sites are unique within the plasmid

of BspMI

(present

three

times).

Among

the 5-bp

recognition sites, only AccI and Ban11 are unique in the polylinker. In particular, the HincII site, which is often listed as a recognition site in the

TABLE

I

Optional

features

pUC18/19

of each plasmid a

Plasmids pDIP

n7ot

transcription

promoter’

terminator’

terminator

(Trnc)

(i-r,)

c

(P,,,,,,)

C

+ +

D

+

+

’ All pDIP plasmids replication

encode

resistance

+

or pUC19.

to Cm(cat)

All have the identical

rIIB which includes a BglII site starting listed are the optional features.

from synthetic

coding

and

region for

in the second codon. The features

oligodeoxynucleotides,

of the mot ORFD promoter

We have that we

to Cm (cat) and the P15A origin (ori)

may be inaccurate

deletion extends at least from

to the BglI site at nt 2657 (Rose, sites and the overall size of the

by a few bp

and have the P15A

’ Albrechtsen et al. (1990). c Studier et al. (1990). the sequence

in pACYC184,

+

to rIIA and rIIB, the T7 $10 promoter,

origin, homology

the MCS of pUC18

The gene that encodes resistance

are derived from pACYC 184. The indicated

plasmid _

sites, present

the HaeII site at nt 1928 in pACYC184 1988). The locations of the restriction

_ _ _

is found three times in these plasmids. the restriction

have eliminated.

T7

B

polylinker,

drawn boxes around

rpoC transcription

A

’ Derived

of the various

(Guild

the mot promoter et al., 1988).

has

1990) we found that this terminator reduced the basal expression of 1ucZ about 15fold (Table II). We compared lucZ expression from the pDIP plasmids with expression from pBC39SD8AUG (Hartz et al., 1990) the source of the ZucZgene that we cloned into pDIP. In this plasmid, kzcZ is under the control of a tuc promoter; the host, D1210 (Sadler et al., 1980) is a luqlq strain; thus

3 TABLE

II

@Gal activity

in transformed

Plasmid ”

fiGa

activity b

Basal

Induced

A.

MC106 1

BL2I(DE3)

none

n.d.

3596

pDIPI8A

n.d.

1934

pDIP19A

n.d.

pDIPl9A

: IacZ

pDIPl9B

: lad

B.

79

42 553 21553

D1210d

01210

none

55 172

measured

IGal

57286

2223

3 See Tabie I and Fig. 1. ’ In order to determine basal

levels attributable

activity in MC1061

(Casadaban

strain that does not contain

BL21(DE3)

(Studier

gene in the integrated expression

DE3 chromosome.

harvested

for SDS-PAGE

c The plasmid

at 37°C.

plasmid,

we

1980), a luc The strain T7 RNAP

We grew the cells in M9 (Belle acids, and assayed

@Gal

At the same time, cells were

(see Fig. 2). n.d., not determined.

pBC39SD8AUG

/acZ gene used in the pDIP : Iad the control

the IPTG-inducible

with O.Z,,” Casamino

2.5 h after induction

to each and Cohen,

the gene for T7 RNAP.

et al., 1990) harbors

et al., I968), suppIemented

+ IPTG

216

1.2

pBC39SDSAUG’

deletion

37017

68 69

: IacZ

+ IPTG

1130

1214

pDIP19C:lacZ pDIP19D

quency by stabilizing the plasmid DNA during phage infection. The T4 chromosome is packaged by a headfilling mechanism, the wt phage having an estimated 3 kb of terminal redundancy (Kim and Davidson, 1974). T4(DIPl9D:~acZ} has little terminal redundancy because the lacZ gene is about that size. Stocks of these phages accumulate more deletions than the corresponding phage that have the compensatory deletion SaA9. The ac gene confers resistance to acri~avine when it is inactivated; moreover, it is in a region of the genome that is largely dispensable (Parma, 1969). The frequency of acR in T4( DIP19D : facZ) stocks is significantly higher than in stocks of T4( DIPI9D > (not shown). Moreover, the /neZ gene is itself occasionally deteted.

bacteria

(Hartz

et al., 1990) is the source of the

constructs.

It has the la&? gene under

of the TUCpromoter.

’ D1210 is a lad* strain

(Sadler

et al., 1980).

uninduced levels are minimal. Basal expression in MC106l[pDIP19A: ZacZ] (which has the highest basal expression among the pDIP plasmids) is about the same as the expression from Dl2lO~p~C39SD8AUG] without IPTG. (d) DIP phage: recombination into T4 The pDIP system uses recombination between r11 sequences resident in the plasmid and rI1 sequences in the phage to incorporate cloned sequences into the T4 chromosome. Fig. 3 shows the procedure for ‘lifting-out’ plasmid sequence into phage. The frequency of the ‘lift-out’ is in part a function of the size of the insert. For instance, pDIP19D : IacZ yields about 10m6 r+ recombinants, while the same plasmid without any insert yields about 50-times more r + recombinants. The ‘lift-out’ frequency can be stimulated about ten-fold, irrespective of the size of the insert, by including Sa49 in thegenetic background of the phage. Sad9 deletes the denB gene that encodes a nuclease that degrades cyto&e-containing DNA (Vetter and Sadowski, 1974); the deletion probably increases the ‘lift-out’ fre-

(e) Expression of ZucZ in DIP phage We constructed T4(DIP19D: facZ) strains with various genetic ba~k~ounds and assayed fiGa activity produced during infection. Table III shows the results of those experiments. In the wt phage, rZIB is transcribed under the control of both early and middle promoters that are located both within and upstream of the t-1%9gene (Guild et al., 1988; Daegelen and Brody, 1990). In the absence of T7 RNAP, the 1acZ gene is under the control of these promoters. In the wt background, use of the T7 promoter increases @Gal activity over lo-fold. Since T4 DNA contains glucosylated hydroxymethyl cytosine (HMC) and G-C pairs occupy several of the highly conserved positions of the T7 promoter (Dunn and Studier, 1983; Schneider and Stormo, 1989), we inhibited the hydroxymethylation of cytosine by adding mutations in genes 42 and 56 (Mathews and Allen, 1983). T4 phages mutant in these genes make DNA that contains unsubstituted cytosine. This DNA is an appropriate substrate for T7 RNAP and the yield of BGal improves dr~atic~ly. Table III shows that optimum expression is found when an amber mutation in gene 46 is added to the 42-56- background. Gene 46 encodes an exonuclease that is required for recombination in T4 (Warner and Snustad, 1983); presumably lack of gp46 stabilizes the DNA substrate for T7 RNAP. We were unable to increase the yield of @Gal by further limiting the amount of middle or late transcription from T4 promoters. We used the antibiotic Rif to restrict T4 transcription, but not transcription catalyzed by T7 RNAP, which is insensitive to Rif (Studier et al., 1990). We found that PGal production in E. co/i infected with T4(DIP19D:IacZ)SaA9 42-56-46is completely insensitive to the addition of Rif at any time after 5 min after infection. In the conditions used, DNA synthesis is expected to begin 6 min after infection. Under these same conditions, BGaI activity increases for at least 2 h. It seems likely that little or no middle and late transcription takes

,QGal

/3Gal

I234567 Fig. 2. Expression plasmids,

of the IucZ reporter

were grown as described

SDS-PAGE

gene. Panel A: Expression

in Table II, footnote

were (1) none; (2) pDIP18A;

is shown, The plasmids Panel B: BL21(DE3),

I

grown

as above, was infected

in induced

(3) pDIP19A;

(4) pDIP19A:

with an moi of 5 at

as above. The infecting phage were (l), T4( DIPl9D)Sad9

to rlIR in strains

containing

Sad9;

~52 is the product

transformed

ceils. BLZl(DE3)

for 0.1 Y’ SDS - 10% PAGE

b, and harvested

(no T7 RNAP) wt

wt

42-56

no HMC

deletion

infect a celi con-

phage and plasmid

can

901 8201 17665

no HMC, lacks exonuclease iacks exonuciease

46-

‘I We grew the indicated

riI

70

(T7 RNAP induced)

46

between

cells @Gal activity h

wt 42‘56

: IacZ > SaA9-infected

T4 mutations”

Il. M~l~l[pARl219]

of the DIP region into phage. When

in T4 (DIP19D

Phenotype

I. MC1061

recombination

gel

: 1acZ.

HI

wt

taining a pDIP plasmid,

(7) pDIP19D

of gene 52.

Genotype

a smalt, intercistronic

/acZ;

pDlP

blue-stained

1h after induction. At 2.5 h after infection, infected cells were prepared for ; (2),T4(DIP19D: lacZ)SadP 42-56-46Gene 52 is adjacent

Additional

containing

or not with various

1970). A Coomassie

42-SK46-

Cloned gene expression

Fig. 3. The lift-out: recombination

cells, transformed

(Laemmli,

IacZ; (5) pDIP19B : IacZ; (6) pDIP19C:

TABLE

phage

2

bacteria

1444 --___I

in M9 (Belle et al., 1968) supplemented

with 0.2”/, Casamino acids. MC1061 (Casadaban and Cohen, 1980) is a non-suppressing luc deletion strain; pAR1219 provides the gene for T7 RNAP (Davanlooet

al., 1984). We infected with a multiplicity

(moi) of about 5 phage per cell, when the cells had reached

ofinfection

an A,,

of 0.35.

yield r + recombinants that have incorporated the DIP cloning region into phage. We transformed CR63 with the pDIP plasmid and infected these

In the case of MCl061[pAR1219), we induced the expression of T7 RNAP at least an hour prior to infection. At 2 h after infection, we used

cells with A26 (rII-

lysis from without to permeabilize the cell for the fiGa assays (Arvidson et al., 1991). h BGal activity is expressed in Miller units (Miller. 1972).

) phage

at a moi of about

one phage per cell. The

progeny that can grow on CR63(2) have incorporated the DIP region from the plasmid. E. coii CR63 is described in Signer et al. (1965).

5 place during the T4(DIP19D:lacZ)Sad9 42-56-46infection of E. coli preinduced for T7 RNAP. In agreement with this conclusion is our finding that mutational inactivation of middle and late transcription does not alter the yield of PGal (data not shown). Addition of Rif prior to 5 min reduces the expression of lacZ (data not shown). We have not pursued the dynamics of this process further. (f) The mot promoter Fig. 2A shows that BL21(DE3) [pDIP18A] (lane 2)produces only a small fraction of rIIB ’ fragment relative to the amount produced by BL21(DE3) [pDIP19A] (lane 3). Since the plasmids should be transcriptionally equivalent, it seems likely that the defect in BL21(DE3) [pDIP18A] is translational. Presumably the mRNA with the polylinker in this orientation can form a structure that occludes the RBS. We inserted a middle (mot) promoter before the rZIB gene so that expression of rZZB in phage derived from these plasmids would be independent of whatever is upstream. We chose to use a mot promoter because the mot promoter is largely silent in the uninfected cell (Guild et al., 1988). (g) Inhibition of proteolysis We cloned the IacZ variant X90, which encodes an unstable protein, in pDIP19D and recombined the gene into phage. When resident in HMS174(DE3), and E. coli K-12 strain with an inducible T7 RNAP (Studier et al., 1990) the X90 protein is highly unstable, with a half life of about 10 min, in accordance with an earlier report (Goldschmidt, 1970). When the X90 protein is produced HMS 174(DE3) by of infection during T4( DIP19D: lacZX90)Sad942-56-46-, the sameprotein is completely stable for at least 1 h (not shown), indicating that T4 inhibition of proteolysis occurs in overproducing conditions.

BGal is overproduced less during T4 ( DIP 19D : 1acZ ) Sad9 42 -56 -46 _ infection than from the corresponding plasmid. On the other hand, Unnithan et al. (1990) found comparable yields of RegA from both phage and plasmid systems. (2) The issue of proteolysis We have shown that T4 inhibits proteolysis even under overproducing conditions. However, the unstable protein that we chose to assay is also stabilized (not shown) in BL21(DE3) [pDIP19D : lacZX90], a B strain deficient in the fan protease (Studier et al., 1990). Overproduction from uninfected, induced cells is probably technically simpler than overproduction from phage-infected cells. We suspect that there are unstable proteins that are stabilized only by T4 infection, and that these new vectors will be important in such cases. (3) The issue of toxicity As noted, the basal level of expression is very low in MC1061[pDIP18/19D], thus allowing the stable cloning of genes that encode relatively toxic proteins. For instance, the bacteriophage T4 regA gene encodes a translational repressor that is quite toxic to the uninfected cell (Miller et al., 1987). When pDIP19A: regA is transformed into MC1061, the plasmid is completely stable. When Unnithan et al. (1990) infected MC106l[pDIP19A: regA] with DE3 (which provides T7RNAP, Studier et al., 1990) RegA became the major protein in the cell. If plasmid-based expression results in cell death before the accumulation of adequate protein, it would be worth trying expression from T4( DIP)-infected cells. The physiology of the uninfected cell is very different from that of the infected cell; the gene product may not be toxic in the latter case.

(h) Conclusions (I) Maximization of gene expression Our results suggest that overexpressing one gene limits the expression of another. For example, only pDIP 18D and pDIP19D have the T7 transcription terminator to limit transcription to the gene of interest, and pDlP19D yields the highest level of PGal (pDIP18D was not assayed). Thus we recommend the use of pDIP18/19D or T4(DIP18/19D) whenever possible. Moreover, eliminating non-essential material from the transcript that encodes the gene of interest is a useful strategy for any cloning project whose end is to maximize expression. During T4 infection, utilization of the T7 promoter by T7 RNAP can be maximized by providing the enzyme with intact, cytosine-containing DNA. We have achieved this by eliminating the T4 pathway that puts HMC into the chromosome and by inactivating a Tsencoded exonuclease.

ACKNOWLEDGEMENTS

This work was supported by NIH Grant GM 19963-18 and NSF Grant BCS-8912259. We thank Hans Huber for determining the 5’ end of the PmO, message. We thank CL. Squires, J. Gott, D. Shub and F.W. Studier for strains. We thank C.A. Tuerk for pUC18 DNA and Jon Binkley for making the oligodeoxynucleotides. We are grateful that Ann Zeeh, Judy Ruckman, and Dennis Arvidson made results available prior to publication.

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In our report we have cited the results of A. Zeeh concerning stabilization of an X90 variant of /IGal during T4 infection. A paper by A. Zeeh, D.A. Shub, L.D. Simon, and K. Skorupsky concerning these results will be published in Gene.