Proc. Nat!. Acad. Sci. USA Vol. 76, No. 11, pp. 5596-5600, November 1979 Biochemistry
Synthesis of simian virus 40 t antigen in Escherichia coli (recombinant plasmid/hybrid ribosome binding site/eukaryotic gene expression/amino acid sequence analysis)
THOMAS M. ROBERTS*, ILAN BIKELt, R. ROGERS YOCUM*, DAVID M. LIVINGSTONt, AND MARK PTASHNE* *The Biological Laboratories, Harvard University, Cambridge, Massachusetts 02138; and tSidney Farber Cancer Institute and the Departments of Medicine, Peter Bent Brigham Hospital and Harvard Medical School, Boston, Massachusetts 02115
Contributed by Mark Ptashne, August 22,1979 Plasmids are constructed by using recombiABSTRACT nation in vitro according to Roberts, T. M., Kacich, R. & Ptashne, M. (1979) Proc. Nat]. Acad. Sci. USA 76,760-764 in which the t antigen gene of simian virus 40 is fused to a promoter of the Escherichia coli lac operon. In the fusions, transcription commences at the lac promoter, and, in some of the fusions, translation begins at the ATG initiator codon of the t gene. This translation is directed most efficiently by those plasmids in which the lac sequences abut the t gene such that a hybrid ribosome binding is encoded. In this case, the Shine-Dalgamo sequence is of lac origin but the ATG derives from the t gene. Translation from this initiator codon is greatly decreased if the lac sequences are separated from the ATG by 17 base pairs and is abolished if the AT of this triplet is deleted. Cells bearing the productive fusions synthesize a 20,000-dalton protein with t antigenic determinants. This protein has an isoelectric point(s) indistinguishable from that of t antigen isolated from simian virus 40-transformed cells. Moreover, a partial sequence of the amino-terminal region of the bacterial product is that predicted for authentic t antigen. We conclude that these bacteria are producing a protein, the sequence of which is identical to that of authentic t antigen unfused to other polypeptides.
The T antigens of simian virus 40 (SV40) are virus-encoded proteins that play a central role in the process of virus-induced neoplastic transformation (1-8). One of these proteins, the t antigen (t), is-a 174-amino acid polypeptide whose coding sequence is known. Unlike the related protein SV40 T antigen (T), it is cytoplasmic and does not bind to DNA in vitro (ref. 1; J. Griffin and D. Livingston, unpublished results). Analysis of t function in vitro has been limited by the fact that it is difficult to purify from SV40-infected cells. The DNA encoding t has no intervening sequences and, if supplied the appropriate cisacting control sequences, its expression in a bacterium should produce a polypeptide identical in sequence to t. In this paper, we describe the construction of plasmids that direct the synthesis of apparently authentic SV40 t in bacterial cells. The essential feature of the construction is the fusion of the t gene to an Escherichia coli DNA fragment bearing a promoter and a so-called Shine-Dalgarno (SD) sequence (9-11), both from the lac operon. The former insures efficient transcription of the t gene. The latter, when positioned at the appropriate distance from the ATG signaling the translational start, directs binding of the mRNA to ribosomes and proper initiation of translation. We refer to such a regulatory sequence as a "hybrid ribosome binding site". The product of translation is not a bacterial-eukaryotic fusion protein. Rather, our experiments strongly suggest that, as expected, it has the primary structure of t. The approach we use to position the DNA fragment containing the bacterial control sequences is based on the The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact.
principle and methods utilized to maximize the production of two regulatory proteins encoded by phage X, repressor and cro protein (12, 13). MATERIALS AND METHODS Animal Cells and Viruses. The SV80 strain of SV40-transformed human fibroblasts (14) served as a source of authentic t. SV40, strain 777, was grown and its DNA was isolated as described (15). Protein was labeled in vivo with [35S]methionine (specific activity, 400-600 Ci/mmol; 1 Ci = 3.7 X 1010 becquerels) at a concentration of 50-100 ,uCi/ml as described (16). Bacterial Cells and Radiolabeling of Protein. E. coli strains MM294 (endo I-, B-, rk-, mk+) and RB113.(recA13, uvrA6, thi-1, thri, leuB6, proA2, argE3, his-4, mtl-1, xyl-5, tsx33, strA31, gal2, aral4, lacYl) were used. Labeling of total cell protein was carried out in MM294 by using Na5SO4 under the condition of Roberts and Roberts (17). Plasmid-coded proteins were labeled in RB113 by using [35S]methionine under the conditions of Sancar et al. (18) as modified by R. Brent (unpublished), the 'maxicell technique'. This technique specifically labels with radioisotope plasmid-coded protein. A typical experiment is performed by lightly UV irradiating recA - uvrA cells bearing the plasmid of interest. The cells are then incubated in nutrient medium for 3 hr at- which time cycloserine is added to 100 ,Ag/ml. The incubation is then continued for 10-14 hr during which time most of the host chromosomal (but not plasmid) DNA is destroyed. Finally, the cells are washed in sulfur-free medium and incubated in the presence of [35S]methionine for another 2 hr. For details, see Sancar et al. (18). Plasmid Construction and Analysis. Enzymes and techniques for plasmid construction and analysis have been reported (12, 13, 19). All plasmids were constructed and propagated under conditions conforming to the standards outlined in the National Institutes of Health Guidelines for Recombinant DNA Research. T Antigen Immunoprecipitation and Gel Electrophoresis. Total cell protein from SV80 cells was labeled with [-5S]methionine, extracted, and immunoprecipitated either with serum from hamsters bearing SV40-induced tumors or with serum from tumor-free animals as described (16). For the immunoprecipitation of t, polypeptides from E. coli extracts were prepared and immunoprecipitated in the presence of an excess of unlabeled E. coli 294 extract as described by Roberts and Roberts (17). Sodium dodecyl sulfate/polyacrylamide gel electrophoresis was as described by Laemmli (20). After fixation, staining, and destaining (where indicated), gels were Abbreviations: SV40, simian virus 40; kd, kilodaltons; SD, ShineDalgarno; t, t antigen; T, T antigen. 5596
Proc. Natl. Acad. Sci. USA 76 (1979)
Biochemistry: Roberts et al. a)
M IMPO ff/ nM7 Xe
5597
A2ns M MOBMom
a =m
Ligate
pBR322
RI
Ac
RI
\"
Synthesis of simian virus 40 t antigen in Escherichia coli (recombinant plasmid/hybrid ribosome binding site/eukaryotic gene expression/amino acid sequence analysis)
THOMAS M. ROBERTS*, ILAN BIKELt, R. ROGERS YOCUM*, DAVID M. LIVINGSTONt, AND MARK PTASHNE* *The Biological Laboratories, Harvard University, Cambridge, Massachusetts 02138; and tSidney Farber Cancer Institute and the Departments of Medicine, Peter Bent Brigham Hospital and Harvard Medical School, Boston, Massachusetts 02115
Contributed by Mark Ptashne, August 22,1979 Plasmids are constructed by using recombiABSTRACT nation in vitro according to Roberts, T. M., Kacich, R. & Ptashne, M. (1979) Proc. Nat]. Acad. Sci. USA 76,760-764 in which the t antigen gene of simian virus 40 is fused to a promoter of the Escherichia coli lac operon. In the fusions, transcription commences at the lac promoter, and, in some of the fusions, translation begins at the ATG initiator codon of the t gene. This translation is directed most efficiently by those plasmids in which the lac sequences abut the t gene such that a hybrid ribosome binding is encoded. In this case, the Shine-Dalgamo sequence is of lac origin but the ATG derives from the t gene. Translation from this initiator codon is greatly decreased if the lac sequences are separated from the ATG by 17 base pairs and is abolished if the AT of this triplet is deleted. Cells bearing the productive fusions synthesize a 20,000-dalton protein with t antigenic determinants. This protein has an isoelectric point(s) indistinguishable from that of t antigen isolated from simian virus 40-transformed cells. Moreover, a partial sequence of the amino-terminal region of the bacterial product is that predicted for authentic t antigen. We conclude that these bacteria are producing a protein, the sequence of which is identical to that of authentic t antigen unfused to other polypeptides.
The T antigens of simian virus 40 (SV40) are virus-encoded proteins that play a central role in the process of virus-induced neoplastic transformation (1-8). One of these proteins, the t antigen (t), is-a 174-amino acid polypeptide whose coding sequence is known. Unlike the related protein SV40 T antigen (T), it is cytoplasmic and does not bind to DNA in vitro (ref. 1; J. Griffin and D. Livingston, unpublished results). Analysis of t function in vitro has been limited by the fact that it is difficult to purify from SV40-infected cells. The DNA encoding t has no intervening sequences and, if supplied the appropriate cisacting control sequences, its expression in a bacterium should produce a polypeptide identical in sequence to t. In this paper, we describe the construction of plasmids that direct the synthesis of apparently authentic SV40 t in bacterial cells. The essential feature of the construction is the fusion of the t gene to an Escherichia coli DNA fragment bearing a promoter and a so-called Shine-Dalgarno (SD) sequence (9-11), both from the lac operon. The former insures efficient transcription of the t gene. The latter, when positioned at the appropriate distance from the ATG signaling the translational start, directs binding of the mRNA to ribosomes and proper initiation of translation. We refer to such a regulatory sequence as a "hybrid ribosome binding site". The product of translation is not a bacterial-eukaryotic fusion protein. Rather, our experiments strongly suggest that, as expected, it has the primary structure of t. The approach we use to position the DNA fragment containing the bacterial control sequences is based on the The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact.
principle and methods utilized to maximize the production of two regulatory proteins encoded by phage X, repressor and cro protein (12, 13). MATERIALS AND METHODS Animal Cells and Viruses. The SV80 strain of SV40-transformed human fibroblasts (14) served as a source of authentic t. SV40, strain 777, was grown and its DNA was isolated as described (15). Protein was labeled in vivo with [35S]methionine (specific activity, 400-600 Ci/mmol; 1 Ci = 3.7 X 1010 becquerels) at a concentration of 50-100 ,uCi/ml as described (16). Bacterial Cells and Radiolabeling of Protein. E. coli strains MM294 (endo I-, B-, rk-, mk+) and RB113.(recA13, uvrA6, thi-1, thri, leuB6, proA2, argE3, his-4, mtl-1, xyl-5, tsx33, strA31, gal2, aral4, lacYl) were used. Labeling of total cell protein was carried out in MM294 by using Na5SO4 under the condition of Roberts and Roberts (17). Plasmid-coded proteins were labeled in RB113 by using [35S]methionine under the conditions of Sancar et al. (18) as modified by R. Brent (unpublished), the 'maxicell technique'. This technique specifically labels with radioisotope plasmid-coded protein. A typical experiment is performed by lightly UV irradiating recA - uvrA cells bearing the plasmid of interest. The cells are then incubated in nutrient medium for 3 hr at- which time cycloserine is added to 100 ,Ag/ml. The incubation is then continued for 10-14 hr during which time most of the host chromosomal (but not plasmid) DNA is destroyed. Finally, the cells are washed in sulfur-free medium and incubated in the presence of [35S]methionine for another 2 hr. For details, see Sancar et al. (18). Plasmid Construction and Analysis. Enzymes and techniques for plasmid construction and analysis have been reported (12, 13, 19). All plasmids were constructed and propagated under conditions conforming to the standards outlined in the National Institutes of Health Guidelines for Recombinant DNA Research. T Antigen Immunoprecipitation and Gel Electrophoresis. Total cell protein from SV80 cells was labeled with [-5S]methionine, extracted, and immunoprecipitated either with serum from hamsters bearing SV40-induced tumors or with serum from tumor-free animals as described (16). For the immunoprecipitation of t, polypeptides from E. coli extracts were prepared and immunoprecipitated in the presence of an excess of unlabeled E. coli 294 extract as described by Roberts and Roberts (17). Sodium dodecyl sulfate/polyacrylamide gel electrophoresis was as described by Laemmli (20). After fixation, staining, and destaining (where indicated), gels were Abbreviations: SV40, simian virus 40; kd, kilodaltons; SD, ShineDalgarno; t, t antigen; T, T antigen. 5596
Proc. Natl. Acad. Sci. USA 76 (1979)
Biochemistry: Roberts et al. a)
M IMPO ff/ nM7 Xe
5597
A2ns M MOBMom
a =m
Ligate
pBR322
RI
Ac
RI
\"
