Phage RNA polymerase vectors that allow efficient gene expression in both prokaryotic and eukaryotic cells

Gene, 164 (1995) 75-79 © 1995 Elsevier Science B.V. All fights reserved. 0378-1119/95/$09.50

75

GENE 09192

Phage RNA polymerase vectors that allow efficient gene expression in both prokaryotic and eukaryotic cells (Translation; internal ribosome entry site; ribosome-binding site; picornavirus; reinitiation)

Biao He, William T. McAllister and Russell K. Durbin Morse Institute for Molecular Genetics, Department of Microbiology and Immunology, SUN Y Health Science Center at Brooklyn, Brooklyn, N Y 11203-2098, USA

Received by F. Barany: 6 March 1995; Accepted: 8 May 1995; Received at publishers: 11 July 1995

SUMMARY

We have developed expression vectors that direct the synthesis of proteins from a common set of signals in both prokaryotic and eukaryotic cells. To allow transcription from a common promoter the vectors rely upon a phage RNA polymerase (RNAP). To direct initiation of translation to the same start codon the vectors utilize an internal ribosome entry site (IRES) from encephalomyocarditis virus (EMCV) that has been modified to include a prokaryotic ribosomebinding site (RBS) at an appropriate distance upstream from the desired start codon. These vectors provide levels of expression in eukaryotic cells that exceed those of a conventional RNAP-II-based system by 7-fold, and expression in bacterial cells at levels comparable to other phage RNAP-based systems. Inclusion of a lac repressor and a phage promoter/lac operator fusion element allows tight regulation. Cotransfection of eukaryotic cells with the expression vector and a vector that encodes the phage RNAP provides high-level transient expression without the need to construct specialized stable cell lines.

INTRODUCTION

For a variety of purposes, it would be useful to have vectors that direct expression of a target gene in both prokaryotic and eukaryotic cells; however, the signals that regulate transcription and translation in these two cell types are quite different. In prior work it was demonstrated that phage RNAPs can function in both types of cell, allowing efficient transcription of target genes from

Correspondence to: Dr. R.K. Durbin, Department of Microbiology and Immunology, SUNY Health Science Center at Brooklyn, 450 Clarkson Avenue, Brooklyn, NY 11203-2098, USA. Tel. (1-718) 270-1238; Fax ( 1-718) 270-2656; e-mail: [email protected]

Abbreviations: aa, amino acid(s); bp, base pair(s); CAT, chloramphenicol acetyltransferase; cat, gene encoding CAT; EMCV, encephalomyocarditis virus; IRES, internal ribosome entry site; IPTG, isopropyl-13o-thiogalactopyranoside; kb, kilobase(s) or 1000 bp; nt, nucleotide(s); ORF, open reading frame; Pollk, Klenow large fragment of E. coli DNA polymerase; RBS, ribosome-binding site; RNAP, RNA polymerase; RSV, Rous sarcoma virus; SEAP, secreted alkaline phosphatase. SSD1 0378-1119(95)00475-0

a common promoter (Fuerst et al., 1986; Deuschle et al., 1989; Studier et al., 1990). To allow translation of the phage transcripts in both cell types, we constructed a vector which incorporates a modified internal ribosome entry site (IRES) from encephalomyocarditis virus (EMCV) that circumvents the requirement for capped mRNA in mammalian cells (Jang et al., 1989; Zhou et al., 1990; Elroy-Stein and Moss, 1990), and which includes a bacterial ribosome-binding site (RBS; Shine and Dalgarno, 1974) that directs translation to the same start codon in prokaryotic cells.

EXPERIMENTAL AND DISCUSSION

(a) Modifications to the 3' end of the IRES that retain activity The start codon in the EMCV-IRES is predicted to be within a stable stem-loop structure, a feature that may be critical to its function (Jang et al., 1988; 1989; Duke

76 et al., 1992). Due to a concern that manipulation of this region might affect IRES function, we had earlier used the EMCV-IRES to direct expression of a cat gene by inserting it into the O R F downstream from the IRES start codon (Zhou et al., 1990). This resulted in the production of a fusion protein in which five aa from the IRES element and twelve aa from an intervening polylinker sequence prefaced the cat sequence (see pYZ29, Fig. 1). It has since been noted that some variability at the 3'

P

end of the IRES is tolerated in naturally occurring strains of EMCV without abolishing the ability of the IRES to promote internal initiation of translation (Duke et al., 1992). Moss and co-workers took advantage of a strain of EMCV that contains an NcoI site at the start codon to replace the region downstream with other coding information, and found that this construct allowed expression of the target gene in a cap-independent manner (Moss et al., 1990; Elroy-Stein and Moss, 1990). As shown in Table I, a similar construct (pBH30) in

EMCV-IRES

cat

:i ~/~i~ii~i~~ ~!!~:.:.:+:.:.:;.:~.i.i~-~.i.i.l.i.!.i.i.~.~ ...............................~g!"~:~:~:::i:i:i:i::~:x'i Plasmids

Promoter

pYZ29

T3

GAT TAT ATG GCC ACA ACC ATG AGC "FIGGCG AGA TIT TCA GGA GCT AAG GAA GCT AAA ATG GAG AAA AAA Met Ala Thr Thr Met Ser Leu Ala Arg Phe Ser Gly Ala Lys Glu A|a Lys Met Glu Lys Lys

pBH31

T7

GAT TAT ATG GCC ACA ACC ATG AGC TrG GCG AGA TIT TCA GGA GCT AAG GAA GCT AAAATG GAG AAA AAA Met Ala Thr Thr Met Ser Leu Ala Arg Phe Set Gly Ala Lys Glu Ala Lys Met Glu Lys Lys

pRKD294

T7

GAT AAT ACC ATG GGA ArT CCC CGG GGA GCT CAC T A G C A T G GAG AAA AAA Met Gly Ile Pro Arg Gly Ala His Stop Met Glu Lys Lys

pBH30

T7

GAT AAT ACC A T G GAG AAA AAA

Met Glu Lys Lys

pBH32

T7

GAT ATTAC GA ATT CCC CGG GGA GCT CA TAG TGC A T G GAG AAA AAA

Met Glu Lys Lys

pBH82

pBH84

T7

T7/lacO

~..~,T..gk~TA

TAA CC A T G GAG AAA AAA Met Glu Lys Lys

GAT AAT AAG GAG GTA TAA CC A T G GAG AAA AAA

Met Glu Lys Lys Fig. 1. Structure of cat expression vectors. Expression of the cat gene is under control of a promoter (P) of phage T3 or T7, or a T7 promoter-lac operator (T7/lacO) fusion element, as indicated. The nt sequence of RNA in the junction region between the EMCV-IRES element and the beginning of the cat gene is indicated, as is the predicted aa sequence. Bold-faced underlined sequences are derived from the EMCV-IRES, non-bold sequences are derived from the polylinker originally cloned in pYZ29, italicized sequences are derived from the cat gene (Zhou et al., 1990). Relevant start and stop codons are printed in large type and/or underlined. The RBS that is present in pBH82 is double-underlined. All plasmid manipulations were carried out using standard methods (Sambrook et al., 1989); sequence files are available upon request. The cat gene in pYZ31 (Zhou et al., 1990) was mutagenized by PCR as described by Ho et al. (1989), using primers A-D as follows: A, 5'-AAGGGTCGACCCATGGAGAAAAAAATCACTGG; B, 5'-CCCGTTTTCACTATGGGCAAATATTATACGCAAGGC; C, 5'-GCCCATAGTGAAAACGGGGGCGAAG; D, 5'-GGGGGGATCCGACGTCAGACATGATAAGATACATTG. The resulting fragment, containing a cat gene in which the starting AUG is within a NcoI site and the internal NcoI site is eliminated, was subcloned into pBR322 to make pBH18, pBH30 was constructed by subcloning a 750-bp Ncol-Sau3A fragment that contains the modified cat gene into pTM1 (Moss et al., 1990); this plasmid is essentially the same as pT7EMCAT (Elroy-Stein and Moss, 1990). To make pRKD294, the modified cat gene fragment was repaired with Pollk and cloned between the Pollk-repaired SpeI and BamHI sites of pTM1. pBH31 was made by replacing the KpnI-EcoRI fragment of pRKD294 with the corresponding fragment from pBSECAT (Jang et al., 1989). pBH32 was constructed by digesting pRKD294 with NcoI, followed by mung bean nuclease treatment and ligation, destroying the start codon at the 3' end of the IRES. pBH82 was made by replacing the KpnI-BamHI fragment of pBH56 with the corresponding fragment of pTM1. pRKD324 was constructed by inserting the EcoRV fragment from pAR1219 (Studier et al., 1990) into the Pollk-repaired HindlII site of pDMI.1 (Deuschle et al., 1989). pRSVSEAP (Berger et al., 1988) was a gift from Dr. Joel Berger.

77

TABLEI CAT andSEAPactivityintrans~cted mammaliancells"

Plasmid: IPTG:

pBH82 -

+

-

pT7-CAT +

92

Plasmidb

66

Cells

45

BHK (T7 RNAP)c

BHK

CAT

SEAP

CAT

SEAP

0 8.0 x 103 + 79 2.9 x 103_+14 2.6 x 104 _+308 2.2 x 104 _+166 6.5 x 103 _+12 2.2 x 104 _+145 1.5 _+0.1 0.7 4-0.3

0 0.50 0.40 0.45 0.45 0.45 0.45 0.90 1.00

0 4.8 2.9 x 103_+216 0.4 13.2 1.8 1.1 2.5 1.9

0 1.30 0.55 1.30 1.40 1.22 1.32 1.05 0.98

30 CAT

None pRKD294 PRSVCAT pBH30 pBH31 pPH32 pBH82 pTM1 pUC18

" Duplicate 35-ram dishes of cells at approx, 70% confluence in Dulbecco's modified Eagle's medium (Gibco) supplemented with 5% fetal calf serum were transfected with 1 I~g of the indicated plasmid and 1 gg of pRSVSEAP using Lipofectamine cationic liposomes (BRL). Where necessary, total DNA was maintained at 2 gg/dish by the addition of pUCI8. The medium was changed after 9-h exposure to DNA. Cell lysates were prepared 30 h after transfection, and assays were performed for CAT activity (Neumann et al., 1987) or for SEAP (Berger et al., 1988). In cases where the CAT activity was extremely high (e.g., pBH30, pBH31 and pBH82) the lysates were diluted a further 10-fold to remain within the linear range of the assay; the units reported reflect this dilution. SEAP activities are expressed as the increase of A415 nm per 100 rain. In most cases, the level of SEAP activity was sufficiently high that the reactions were not carried out for the full 100-min interval. b Plasmids are described in Fig. 1 or in the text. ° BHK-T7RNAP was constructed by co-transfection of BHK cells with pYZ3 and pTG76, as previously described (Zhou et al., 1990; Giordano and McAllister, 1990). which a m o d i f i e d cat gene has been inserted into this N c o I site also directs high levels of expression in cell lines ( B H K - T 7 R N A P ) t h a t s t a b l y express T7 R N A P . To e x p l o r e w h e t h e r the distance from the u p s t r e a m region of the I R E S to the start c o d o n is critical, we a b l a t e d the start c o d o n of the E M C V - I R E S by m a n i p u l a tion of the N c o I site a n d m o v e d the start c o d o n to a new l o c a t i o n a b o u t 24 nt d o w n s t r e a m b y insertion of unrelated sequences (see pBH32, Fig. 1); this c o n s t r u c t was also active in B H K - T 7 R N A P cells (Table I). E x p e r i m e n t s using constructs a n a l o g o u s to p B H 3 0 a n d pBH32, b u t c o n t a i n i n g a T3 r a t h e r t h a n a T7 p r o m o t e r , p r o d u c e d similar results in B H K cells t h a t express T3 R N A P ( d a t a n o t shown).

21

14 STD

1

2

3

4

Fig. 2. Regulated expression of the cat gene in bacterial cells. E. coli BL21(DE3) cells (Studier et al., 1990) which express T7 RNAP from a chromosomal copy of T7 gene 1 under control of the lac promoter, were transfected with pDM 1.1 (a P15A replicon that expresses the lacl ~ gene; Deuschle et al., 1989) and either pBH82 (lanes 1 and 2) or pT7-CAT (GIBCO/BRL; lanes 3 and 4). Cells were grown to an A6o0 rim=0.6, and 0.4 mM IPTG was added as indicated. After 4 h of induction, samples (0.1 ml) of the culture were harvested and analyzed by gel electrophoresis (Giordano et al., 1989). Molecular mass markers (Bio-Rad) are present in the left lane; sizes are indicated in the left margin (in kDa). levels of cat expression are o b s e r v e d u p o n i n d u c t i o n of these cells with I P T G , a n d these levels are c o m p a r a b l e to those described p r e v i o u s l y using o t h e r T3 a n d T7 expression vectors ( G i o r d a n o et al., 1989; D u b e n d o r f f a n d Studier, 1991). Little expression is detected in cells which lack the T7 R N A P - e n c o d i n g gene o r in the absence of induction, i n d i c a t i n g that the target gene is tightly regulated. T h e a c c u r a c y of t r a n s l a t i o n a l initiation in these cells was c o n f i r m e d b y N - t e r m i n a l a a sequencing of CAT p r o t e i n excised from the gel ( d a t a n o t shown). In m a m m a l i a n cells, p B H 8 2 directs high levels of cat expression (Table I), i n d i c a t i n g t h a t the i n t r o d u c t i o n of the bacterial RBS is c o m p a t i b l e with I R E S function in these cells. The o b s e r v e d cat activity is at least 7-fold higher t h a n t h a t o b s e r v e d with p R S V - C A T , in which cat expression is u n d e r c o n t r o l of a s t r o n g R N A P II p r o m o t e r ( G o r m a n et al., 1982). It should be n o t e d that at the same time when the m o d i f i c a t i o n s described a b o v e were i n t r o d u c e d into pBH82, the cat start c o d o n was p l a c e d in the context of a K o z a k (1978) consensus sequence with the i n t e n t i o n that this might e n h a n c e t r a n s l a t i o n a l efficiency in e u k a r y o t i c cells, We do n o t k n o w if this m o d i f i c a t i o n is essential to the efficiency of the system.

(b) Introduction of a bacterial RBS H a v i n g f o u n d that the precise distance of the start c o d o n from the u p s t r e a m region of the I R E S is n o t critical, we then p l a c e d a p r o k a r y o t i c RBS into this region (pBH82, Fig. 1) a n d i n t r o d u c e d this p l a s m i d into E. coli B L 2 1 ( D E 3 ) , in which expression of T7 R N A P is u n d e r c o n t r o l of the lac p r o m o t e r . As s h o w n in Fig. 2, very high

(c) Cotransfection with the T7 RNAP-encoding gene (gene 1) and the target gene In the studies described a b o v e we used stable cell lines that constitutively p r o d u c e T7 R N A P to direct expression of the target gene. However, such cell lines are not r e q u i r e d for expression, as the gene that encodes the

78 RNAP may be introduced simultaneously with the target gene by cotransfection with an appropriate vecto r (Zhou et al., 1990). For example, when B H K cells are transfected with pYZ3 (which expresses T7 gene I under control of an SV40 promoter) along with pBH82 high levels of cat expression are obtained within 24 h (Table II). Co-transfection of B H K - T 7 R N A P cells (which constitutively produce T7 RNAP) with pYZ3 along with pBH82 also stimulates production of CAT above the basal level.

(d) Translation re-initiation vectors Concurrently with the approach described above, we sought alternate methods to direct translation initiation to a desired start codon in eukaryotic cells. It had previously been reported that in mammalian cells, ribosome complexes that are terminated at a stop codon may reinitiate at a nearby downstream start codon (Peabody and Berg, 1986; Adams et al., 1991). To explore the efficiency of translation reinitiation in this system, we placed a stop codon (UAG) into the region between the start codon of the IRES and the start codon of cat (see pRKD294). This manipulation places the start codon of cat out of frame with the IRES start codon so that production of functional CAT protein can only arise by reinitiation. As shown in Table I, pRKD294 directs expression of c a t in cells that produce T7 RNAP (but not in cells that lack the RNAP) at levels that are nearly as high as that provided by pBH31. Although pBH31 directs the synthesis of a fusion protein rather than the authentic CAT protein, the specific activities of similar N-terminal CAT fusion proteins have been found to be indistinguishable from that of the wild-type protein (Polayes, 1994), thus allowing direct comparison of c a t activities in the two extracts. These results demonstrate that translation reinitiation is quite efficient in this system. Based upon this observation, it may be possible to construct multi-cistronic expression TABLE II CAT and SEAP activitiesin cells that express the T7 RNAP-encoding gene stably (BHK-T7RNAP)or transiently (BHK)a Plasmidsb

Cells

(e) Conclusions In this work, we have manipulated the EMCV-IRES to direct initiation of translation to the same start codon in both prokaryotic and eukaryotic cells, and have placed this element under the control of a phage promoter to allow efficient transcription in both cell types (see pBH82, Fig. 1). Subsequent modifications to this vector to enhance its utility include the introduction of the lac operator just downstream from the T7 promoter to permit more stringent regulation by lac repressor (see pBH84, Fig. 1; Giordano et al., 1989; Dubendorff and Studier, 1991). Although we have not demonstrated regulation of this particular target gene in mammalian cells, similar constructs have previously been shown to be tightly regulated in cell lines that stably produce lac repressor (Deuschle et al., 1989; Zhou et al., 1990). Recently, there have been reports of the use of a T7 RNAP 'autogene' to direct the simultaneous expression of T7 RNAP and a target gene under control of a T7 promoter in mammalian cells (Chen et al., 1994; Deng and Wolff, 1994; Gao et al., 1994). In this approach, the gene that encodes T7 RNAP is itself placed under control of a T7 promoter and introduced into a cell that contains a low level of T7 RNAP (e.g., by delivery of the enzyme using liposomes, or by introduction of an mRNA that encodes T7 RNAP), resulting in very efficient autocatalyric expression of the RNAP gene. As this approach eliminates the need for a nuclear expression phase, a higher frequency of cells in the population that express the protein may be obtained (Chen et al., 1994; Deng and Wolff, 1994; Gao et al., 1994). The use of these vectors would complement and extend the observations reported here, and in preliminary experiments we have confirmed that the use of an autogene stimulates the expression of pBH82 in B H K - T 7 R N A P cells (data not shown).

ACKNOWLEDGEMENTS

BHK-T7RNAP

pRSVCAT (2 I~g) pBH82 (2 ~tg) pBH82 (1 Isg)+pYZ3 (1 Ixg)

vectors that function in both prokaryotic and eukaryotic cells by introducing a prokaryotic RBS into regions that lie just upstream from the re-initiation codon(s).

BHK

CAT

S E A P CAT

SEAP

1422 2908 10254

1.25 1.89 1.09

1.26 2.91 1.84

2003 0 9656

Transfections and enzyme assays were carried out as described in footnotes a and b to Table I. b The cat gene is under control of the RSV promoter in pRSVCATand under control of a T7 promoter in pBH82 (see Fig. 1). pYZ3 encodes T7 RNAP under control of an SV40 promoter (Zhou et al., 1990).

This work was supported by N I H grant GM38147 and by GIBCO/BRL. We are grateful to Ms. Heidi Gartenstein for expert technical assistance and to Joel Jessee (BRL) for encouragement.

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