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Tumor Antigen (Hybrid Trp-Lac Promoter/Actin Cable Effect) ILAN BIKEL*, THOMAS M

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Tumor Antigen (Hybrid Trp-Lac Promoter/Actin Cable Effect) ILAN BIKEL*, THOMAS M Proc. NatL Acad. Sci. USA Vol. 80, pp. 906-910, February 1983 Biochemistry Purification of biologically active simian virus 40 small tumor antigen (hybrid trp-lac promoter/actin cable effect) ILAN BIKEL*, THOMAS M. ROBERTS*, MARILUCI T. BLADON*, ROBERT GREEN*, EGON AMANNt, AND DAVID M. LIVINGSTON* *The Sidney Farber Cancer Institute and The Departments of Medicine and Pathology, Harvard Medical School, Boston, Massachusetts 02115; and tDepartment of Biochemistry and Molecular Biology, Harvard University, Cambridge, Massachusetts 02138 Communicated by Morris Friedkin, October 29, 1982 The simian virus 40 small tumor antigen (t antigen) gene has been were grown in L broth containing ampicillin (40 Ag/ml) to an cloned downstream from a hybrid Escherichia coli trp-lac pro- ODW of0.7 and then incubated for 2 hr with 5 mM isopropyl moter anda suitable ribosome binding site. Abacterial clone (865i) f3-D-thiogalactopyranoside. Radiolabeling of proteins with transformed by such a plasmid (pTR865) expresses this gene and, [3S]methionine was performed as described (31). under optimal conditions, can produce .5% ofits total protein as t Antigen and Protein Assays. t antigen was detected by Na- t antigen. Soluble extracts ofsuch a clone were relatively depleted DodSO4/polyacrylamide slab gel electrophoresis (32) using 4% in t antigen, which was found in the initial pellet fraction. The stacking and 15% separating gels. Marker proteins were bovine protein was recovered from this fraction in a significantly purified serum albumin (Mr 68,000), carbonic anhydrase (29,000), form byextraction with urea-containing buffer. After gel filtration concentrations were deter- of such t antigen-enriched solutions, highly purified protein was and myoglobin (17,000). Protein obtained. When either this fraction (freed ofurea) or NaDodSO4 mined by the method of Lowry et al. (33) with bovine serum gel-purified 865i t antigen (rendered free of detergent) was in- albumin as a standard. jected into untransformed rat cells, dissolution of intracellular Immunoprecipitation. [35S]Methionine-labeled cells were actin cable networks was observed. solubilized and the extracts were immunoprecipitated and elec- trophoresed as described (31). The early region ofsimian virus 40 encodes two products, large Purification of t Antigen. Washed E. coli cells were sus- tumor antigen (T antigen) and small tumor antigen (t antigen), pended (1 g wet weight per 8 ml) in 50 mM Tris-HCI, pH 7.9/ which play an important role in the process of virus-induced 25% sucrose/1% Nonidet P-40/0.5% sodium deoxycholate/2 neoplastic transformation (1-19). t antigen is a 20-kilodalton mM dithiothreitol/5 mM EDTA and sonicated. After centrif- (kDal), nonphosphorylated polypeptide, and most of it can be ugation at 6,000 X g for 30 min at 40C, the pellet, which con- isolated from the cytoplasmic fraction ofinfected cells (20, 21). tained 290% of the cell content oft antigen, was taken as the Its precise biochemical function is unknown, although its pri- starting fraction for further protein purification. This fraction mary structure is characterized by two cysteine clusters which was reextracted twice at 0C by sonicating in 25 mM Tris HCI, also occur in a group ofglycopeptide tropic hormones (22). Fur- pH 8.4/2 mM dithiothreitol (buffer A) containing 2 M NaCl and thermore, repeated, specific co-immunoprecipitation of papo- 2 M urea. After centrifugation, each of these supernatant frac- vavirus t antigen and two cell proteins (56 and 32 kDal) from tions was discarded. Finally, the pellet was suspended in buffer crude cell extracts has been observed (23-25). These results A containing 10 M urea, sonicated, and then centrifuged at 18°C suggest that the antigen can form an intracellular complex with for 30 min at 6,000 x g. This extraction was repeated twice, and these two cell gene products. Finally, there is evidence linking the supernatants were combined. Subsequent purification the synthesis of intact t antigen to the acute disappearance of steps were performed on this fraction (extract A). The yield of intracellular actin cable networks (26, 27), the new appearance t antigen in extract A was found to be independent of the use ofcentriolar structures (28), and, in the presence of T antigen, of lysozyme prior to detergent treatment and sonication. For the promotion ofcell DNA synthesis in untransformed, resting further purification of t antigen, 1 ml of extract A, containing cells (3, 27, 29). How these phenomena result from the func- 0.5-2 mg of protein, was applied to a 2.6 X 40 cm column of tion(s) oft antigen is unknown. Sephacryl S-200 (Pharmacia) equilibrated at 32°C in buffer A This report describes the generation of a rich, prokaryotic containing 10 M urea; 0.8-ml fractions were collected. Fractions source of intact simian virus 40 t antigen and its extensive pu- containing t antigen and 14-kDal t antigen (see Results) were rification in a biologically active form. The availability of such pooled and dialyzed against three changes of 500 vol of buffer a preparation ofthe antigen hopefully will facilitate future anal- A. Both proteins were stored in a soluble form at 0°C in buffer yses of its functions. A at 60-100 ,ug/ml. Purification of tAntigen byNaDodSO4Gel Electrophoresis. MATERIALS AND METHODS t antigen and 14-kDal t antigen (see Results) were purified to simian homogeneity from extract A by preparative NaDodSO4/poly- Growth of Cells and Radiolabeling of Proteins. A Each was eluted- virus 40-transformed cell line, SV80 (30), was grown as de- acrylamide gel electrophoresis (32). protein scribed (31). Escherichia coli W3110 (rK-, mK+, lac iq) a high- from crushed, unfixed, unstained gel strips into 0.2 M ammo- level lac repressor-producing strain, was transformed with nium bicarbonate, pH 8.0/0.4% NaDodSO4 at 320C for 48 hr. pTR865 (see Results). W3110iq/865 (865i) transformed colonies Eluates were lyophilized, and the solid residue was redissolved in water. Each solution was dialyzed at 40C for two 16-hr pe- The publication costs ofthis article were defrayed in part by page charge payment.. This article must therefore be hereby marked "advertise- Abbreviations: t antigen, small tumor antigen; T antigen, large tumor ment" in accordance with 18 U. S.. C. §1734 solely to indicate this fact. antigen; kDal, kilodalton. 906 Downloaded by guest on September 29, 2021 Biochemistry: Bikel et al. Proc. Natl. Acad. Sci. USA 80 (1983) 907 riods, once against 500 vol ofbuffer B (25 mM Tris HCI, pH 7.4/ a relevant segment ofthe E. coli trp operon is cloned in the Cla 0.14 M NaCi) containing 1% cholic acid and once against 500 I site of pBR322. In one of the resulting plasmids, pEA108, vol ofbuffer B containing 0.1% cholic acid. After three further there is a unique Cla I site adjacent to the -35 region of the 16-hr dialyses against 500 vol ofbuffer B containing 0.1% cholic trp promoter. acid, dialysis was performed against three changes of 500 vol In the actual construction (Fig. 1), an Hpa II-EcoRI fragment of buffer B. from pTR436C, bearing the small t antigen gene, ribosome Extraction with Solutions Containing Organic Solvent. Var- binding site, lac operator, and lac promoter through base -21, ious amounts ofextract A (0.3-0.6 mg/ml) were dialyzed against was removed from pTR436C (a modification of pTR436 kindly two changes of 100 vol of (i) 40% 1-propanol/0.25 M urea/0.6 provided by Kai Zinn in which the first Hha I site distal to the M Na acetate, pH 8.1, (ii) 75% 1-propanol/0.25 M Na acetate, t antigen encoding region of pTR436 has been converted to an pH 8.1, or (iii) chloroform/methanol, 1.8:1 (vol/vol), for 16 hr EcoRI site by using a synthetic EcoRI linker) and cloned be- at room temperature. A precipitate formed in each case and was tween the unique Cla I and EcoRI sites of pEA108. In the re- removed by centrifugation at-the end of the dialysis period. sulting plasmid, pTR865, the -35 consensus sequence of ptrp Preparation of Monospecific Anti-t/T Antigen. Two New (cross-hatched area) is joined to the pTR436C sequence. Thus, Zealand White rabbits were each injected, on days 1, 8, and 28, plac is converted to ptac with no modification ofthe messenger with NaDodSO4 gel bands containing 200 pAg of 865i t antigen encoding regions ofthe hybrid gene. This plasmid was used to emulsified in Freund adjuvant. Sera were assayed for [35S]- transform E. coli W31JJOi, a strain that constitutively produces methionine-labeled t/T antigen binding by immunoprecipita- high levels of the lac repressor. A typical colony (865i) grew tion and were absorbed with a sonicated extract ofuntransformed efficiently and synthesized easily detectable amounts of a 20- E. coli W3110iq cells (10 mg of protein per ml). kDal protein (Fig. 2a; compare lanes B and G) which migrated Manual Injection of Various Solutions into Rat-1 Cells and with pTR436c [35S]methionine-labeled t antigen. In addition, Visualization of Actin-Containing Cable Structures. Microin- when a culture of 865i was incubated in the presence of iso- jection of Rat-1 cells growing on glass coverslips and immu- propyl thiogalactoside, the concentration ofthe 20-kDal protein nofluorescent screening of these cells for actin cable networks increased dramatically. were performed as described (19, 26). In all experiments, scor- To extract the 20-kDal protein, isopropyl thiogalactoside-in- ing ofinjected cells was performed under conditions such that duced 865i cells were lysed by sonication in the presence of the analyst was not aware of what had been injected into the dithiothreitol, Nonidet P-40, and deoxycholate.
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