Proc. Nati. Acad. Sci. USA Vol. 86, pp. 2549-2553, April 1989 Biochemistry Transfer of the inducible lac repressor/operator system from Escherichia coli to a vaccinia virus expression vector (mammalian expression vector/transcription regulation/lac repressor/lac operator) THOMAS R. FUERST*, M. PILAR FERNANDEZt, AND BERNARD Mosst Laboratory of Viral Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892 Contributed by Bernard Moss, December 16, 1988 ABSTRACT Cis- and trans-acting elements of the Esche- sequence, which overlaps the start point of transcription of richia coli lac operon were transferred to vaccinia virus and the lac operon. The lac repressor can also bind to allolactose used to regulate gene expression. A recombinant virus that or a nonmetabolizable derivative such as isopropyl f3- constitutively expresses a modified lac repressor gene (lacl) was D-thiogalactoside (IPTG), which then decreases the affinity constructed. We calculated that each infected cell contained =2 of the repressor for lacO. In this manner, IPTG can diminish x 107 active repressor molecules (and 1-2 x 104 copies of the the repression of lac operon transcription, resulting in an vaccinia virus genome). A strong vaccinia-virus late promoter induction of expression. was modified by insertion of the lac operator (lacO) at various Vaccinia virus, like other members of the poxvirus family, positions. The ability of each modified promoter to regulate replicates in the cytoplasm and encodes its own transcrip- expression of (3-galactosidase was tested by transient assays in tional system (13). The RNA polymerase and specific factors cells infected with wild-type or lacI-containing vaccinia virus. for initiation and termination of transcription of the early Placement of the lacO just downstream of the conserved class of genes (14, 15) are packaged within the infectious TAAAT sequence of a late promoter was consistent with a particle. A switch from early-to-late gene expression follows miinimal effect on basal expression and good repressibility, the replication ofthe vaccinia virus genome and involves the whereas basal expression was severely inhibited when IacO synthesis of new transcription factors (ref. 16; C. Wright and overlapped or preceded the TAAAT motif. A single recombi- B.M., unpublished work). To meet our objective, we wished nant vaccinia virus containing Ila and the f-galactosidase gene to insert the lacI gene into the genome of vaccinia virus so under control ofthe optimal bacO promoter was constructed. In that sufficient amounts ofbiologically active repressor would the absence of inducer, cells infected with this double recom- be made and also to place a vaccinia virus promoter under binant virus synthesized little or no detectable (3-galactosidase. control of lacO. In this communication, we describe a Addition of isopropyl fi-D-thiogalactoside restored expression recombinant vaccinia virus that constitutively expresses lac to >20% ofthe unrepressed level. This inducible vector system repressor and can be induced with IPTG to express 3- has potential applications for expression of heterologous and galactosidase. homologous genes. MATERIALS AND METHODS Vaccinia virus has been developed as a vector for expression of heterologous genes (1-3). The availability of plasmid Construction ofPlasmids. Plasmid placl was constructed by transfer vectors containing a vaccinia virus promoter, sites inserting a 1.1-kilobase (kb) pair Bgl II fragment from for insertion of foreign genes, and a variety of markers for pMTlacI (11) into pGS53 (8). The series of recombinant selection and/or screening of recombinant plaques (4-6) has plasmids, placOZ-1, placOZ-2, and placOZ-3, were derived facilitated the use ofthis system. With the appropriate choice from a parent vector designated pMJ11 (received from A. J. of vaccinia virus promoters, inserted genes can be expressed Davison, University of Glasgow). Plasmid pMJ11 contains during the entire growth cycle ofthe virus or only during early the vaccinia virus thymidine kinase (TK) DNA sequence or late phases. Expression of bacteriophage 17 RNA poly- inserted into a pUC-based vector. The TK sequence was merase by vaccinia virus has allowed selective high-level bisected by cleavage with EcoRI into which a lacZ gene was transcription ofgenes that have been engineered to contain a inserted with the following polylinker sequence: 5'- T7 promoter and are present in either a transfected plasmid GGATCC ,GTCGAC ,AAGCTT ,GGTACC ,CTCGAG, (7) or a second coinfected virus (8). Still missing from CATG-3'. The six nucleotide sets correspond to restriction vaccinia virus vectors, however, is the capability of inducing endonucleases BamHI, Sal I, HindIII, Kpn I, and Xho I, specific gene expression on command. In the absence of any respectively. The ATG represents the first codon of the lacZ known naturally inducible vaccinia virus promoter, we con- coding sequence. Plasmid pMJ11 was cleaved sequentially sidered the importation ofone from eukaryotic or prokaryotic with HindIII and Xho I into which a set of oligonucleotides sources. Of the latter, we chose to explore the use of the were inserted. These oligonucleotides reconstructed the operator/repressor system of the Escherichia coli lactose vaccinia virus late promoter, P11, with lacO interposed in operon (9) because it is well characterized, exhibits both three different positions (see Fig. 3). The oligonucleotides stringent repression and high inducibility, and has been used for placOZ-1 were PE1, PE2, PE3, and PE4; the adapted to mammalian cells (10-12). oligonucleotides used for placOZ-2 were PE1, PE2, PE5, and Enzymes encoded within the E. coli lac operon are under PE6; and the oligonucleotides used for placOZ-3 were PE1, the negative control ofa repressor consisting offour identical 38.6-kDa subunits encoded by the lacI gene. The repressor Abbreviations: IPTG, isopropyl P-D-thiogalactoside; TK, thymidine can bind specifically and with high affinity to the lacO kinase; pfu, plaque-forming unit(s); moi, multiplicity of infection. *Present address: Molecular Vaccines Inc., 19 Firstfield Road, Gaithersburg, MD 20878. The publication costs of this article were defrayed in part by page charge tPresent address: Departamento de Biologia Funcional, Facultad de payment. This article must therefore be hereby marked "advertisement" Medicina, Universidad de Oviedo, Oviedo, Spain 33006. in accordance with 18 U.S.C. §1734 solely to indicate this fact. tTo whom reprint requests should be addressed. 2549 Downloaded by guest on October 2, 2021 2550 Biochemistry: Fuerst et al. Proc. NatL Acad. Sci. USA 86 (1989) PE2, PE7, and PE8. Sequences of these oligonucleotides were as follows: A B l 2 3 4 PE1: 5'-AGCTTAAAAATATAGTAGAATTlCATTTTGTT- BamHl IF'T-3' Sal iSmal _ '' ,;.. PE2: 5'-TAGCATAGAAAAAAACAAAATGAAATTCTA- __g CTATATlTfTTA-3'; TK L P TKR ..-..- ... PE3: 7.5 *.: ;: 5'-CTATGCTATAAATAGAATTGTGAGCGCTCA- :: CAATTC-3'; pGS53 \ PE4: 5 '-TCGAGAATTGTGAGCGCTCACAATTCTA- TTT:5A-3'; PE5: 5'-CTATGCTATAAATTGTGAGCGCTCACAA1T-3'; BgIll BgIll PE6: 5'-TCGAAATTGTGAGCGCTCACAATTTA-3'; PE7: 5'-CTATGCTAGAATTGTGAGCGCTCACAATTC- TAAATAC-3'; and ATG TAA PE8: 5'-TCGAGTATTTAGAATTGTGAGCGCTCACAA- lacl fragment TTC-3'. Plasmid placl/OZ-1 was constructed by cleaving placl with Sal Smal Sma I and Sal I and isolating a 1.4-kb DNA fragment containing the lacI gene downstream of the P7.5 promoter. TKL p-ATG TAA TKR This fragment was inserted between the BamHI site made blunt with Klenow polymerase and Sal I site of placOZ-1. placI The promoters and coding sequences of lacI and lacZ were nfection/Transfection positioned in opposite orientation relative to each other. BUdR Selection Isolate TK- Virus RESULTS Construction of a Recombinant Vaccinia Virus That Ex- vlacl presses the lac Gene. A modified lacI gene, in which the natural GUG translation initiation codon was replaced by FIG. 1. Construction of a recombinant vaccinia virus that ex- AUG (11), was presses lacI. (A) A Bgl II fragment containing the lacI gene was inserted into the transfer vector pGS53 (8) so inserted into the unique BamHI site of pGS53 to form placl. In the that it was (i) adjacent to the vaccinia virus P7.5 promoter, latter plasmid lacI is downstream of the vaccinia P7.5 promoter, and which would ensure both early and late expression (17) and the hybrid gene is flanked by the left (TKL) and right (TKR) halves (ii) between the left and right halves of the vaccinia virus TK ofthe vaccinia TK gene. CV-1 cells were infected with vaccinia virus gene (Fig. 1A). The resulting plasmid, placI, was used to and transfected with placl. After 48 hr, the cells were harvested and transfect CV-1 cells that were also infected with vaccinia tested for plaques on TK- cells with 5-bromodeoxyuridine (BUdR). virus. Recombinant virus was then selected by plaque assay Virus plaques were picked and screened by dot-blot hybridization to on TK- cells in the presence of 5-bromodeoxyuridine and laI DNA; the recombinant virus vlacI was then purified. (B) distinguished from spontaneous TK- Immunoblot analysis for detection of lac repressor. CV-1 cells were mutants by dot-blot infected with either recombinant (vlacl) or wild-type vaccinia viruses hybridization to a lacI DNA probe (4). A recombinant virus, at a multiplicity of15 plaque-forming units (pfu) per cell. Cell extracts designated vlacl, was purified, and the correct insertion of were prepared 24 hr after infection, and samples were analyzed by the lac repressor into the TK gene was confirmed by SDS/polyacrylamide gel electrophoresis, blotted to nitrocellulose, Southern blot hybridization. and probed with anti-lac repressor monoclonal antibody B-2, fol- Evidence was obtained that lacI was expressed by the lowed by 1251-labeled protein A. Lanes: 1, 1 ,ug of purified E. coli lac recombinant vaccinia virus. Extracts from cells that were repressor protein; 2, extract from cells infected with vlacl; 3, extract infected with vlacI or wild-type vaccinia virus were dissoci- from cells infected with wild-type virus; and 4, extract from unin- ated with SDS and analyzed by polyacrylamide gel electro- fected cells.