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Biochemistry 1987, 26, 1322-1326
Govil, G., & Hosur, R. V. (1980) Conformation of Biological Molecules; New Results f r o m N M R , Springer-Verlag, Heidelberg. Gronenborn, A. M., & Clore, G. M. (1985) Prog. Nucl. Magn. Reson. Spectrosc. 17, 1-32. Hare, D. R., Wimmer, D. L., Cohn, S. H., Drobny, G., & Reid, B. R. (1983) J . Mol. Biol. 171, 319-336. Hosur, R. V., Wider, G., & Wuthrich, K. (1983) Eur. J . Biochem. 130, 497-508. Hosur, R. V., Ravi Kumar, M., Roy, K. B., Tan, Z., Miles, H. T., & Govil, G. (1985a) Magnetic Resonance in Biology and Medicine, pp 243-260, Tata McGraw-Hill, New Delhi. Hosur, R. V., Chary, K. V. R., Anil Kumar, & Govil, G. (198513) J . Magn. Reson. 62, 123-127. Hosur, R. V., Ravi Kumar, M., Chary, K. V. R., Sheth, A,, Govil, G., & Miles, H. T. (1986) FEBS Lett. 205, 71-76. Jeener, I. (1971) Ampere International Summer School, Basko Polje, Yugoslavia. Maxam, A. M., & Gilbert, W. (1980) Methods Enzymol. 65, 499-560. Munt, N. A., & Kearns, D. R. (1984) Biochemistry 25, 791-796. Ohlendrof, D. H., & Mathews, D. W. (1983) Annu. Rev. Biophys. Bioeng. 12, 259-284.
Ravi Kumar, M., Hosur, R. V., Roy, K. B., Miles, H. T., & Govil, G. (1985) Biochemistry 24, 7703-771 1. Reid, D. G., Salisbury, S. A., Bellard, S., Shakked, Z . , & Williams, D. H. (1983) Biochemistry 22, 2019-2025. Scheek, R. M., Russo, N., Boelens, R., Kaptein, R., & van Boom, J. H. (1983) J . A m . Chem. SOC.105, 2914-2916. Scheek, R. M., Boelens, R., Russo, N., van Boom, J. H., & Kaptein, R. (1984) Biochemistry 23, 1371-1376. Sheth, A., Ravi Kumar, M., Hosur, R. V. Tan, Z., Miles, H. T., & Govil, G. (1987) Biopolymers (in press). Strop, P., Wider, G., & Wuthrich, K. (1983) J . Mol. Biol. 166, 641-665. Tan, Z . , Ikuta, S., Huang, T., Dugaiczyk, A., & Itakura, K. (1982) Cold Spring Harbor Symp. Quant. Biol. 47, 383-391. Wagner, G., & Wuthrich, K. (1982a) J. Mol. Biol. 159, 347-366. Wagner, G., & Wuthrich, K. (1982b) J. Mol. Biol. 160, 334-340. Wagner, G., Anil Kumar, & Wuthrich, K. (1981) Eur. J . Biochem. 114, 375-384. Williamson, M. P., Marion, D., & Wuthrich, K. (1984) J . Mol. Biol. 173, 341-359.
Structure of Human Tumor Necrosis Factor
a!
Derived from Recombinant DNA
Janice M . Davis, Michael A. Narachi, N. Kirby Alton, and Tsutomu Arakawa*
Amgen, Thousand Oaks, California 91320 Received May 2, 1986; Revised Manuscript Received October 2, I986
ABSTRACT: Recombinant D N A derived tumor necrosis factor cy, when expressed at a high level in Escherichia
coli,appeared in the pellet and soluble fractions of disrupted cells. The protein was purified from the pellet fraction by solubilizing it in urea and reducing agent and was refolded into a buffer without these additives. The structure of the protein was identical with that purified from the soluble fraction without exposure to both reducing and denaturing agents, as demonstrated by circular dichroism, gel filtration, and sulfhydryl titration. As a reflection of the structural similarity, both purified proteins showed identical cytolytic activity on mouse L929 cells. The protein was characterized as an essentially nonhelical and P-sheet-rich structure and possibly as a noncovalently associating oligomer. Two cysteine residues form an intrapolypeptide disulfide bond.
x m o r necrosis factor cy (TNF-a)’ was first observed by Carswell et al. (1975) in the serum of endotoxin-treated mice and rabbits that had previously been sensitized with Bacillus Calmette-Guerin. This factor caused hemorrhagic necrosis of various tumors in mice. In addition, it has been shown that TNF-a exhibits cytolytic or cytostatic activities against animal and human transformed cell lines in vitro, but normal cell cultures seem unaffected (Carswell et al., 1975; Ruff & Gifford, 1981; Matthews, 1981, 1982; Hammerstrom, 1982; Helson et al., 1985; Green et al., 1976). The amino acid sequence of human TNF-a has been deduced from cDNA clones (Pennica et al., 1984; Marmenout et al., 1985; Wang et al., 1985; Shirai et al., 1985) and also directly determined
* Author to whom correspondence should be addressed.
from the protein purified from serum-free cell culture supernatants of the HL-60 promyelocytic leukemia cell line induced by 5P-phorbol 12-myristate 13-acetate (Aggarwal et al., 1985). The gene coding for TNF-a has been cloned, and the corresponding protein has been expressed in Escherichia coli. It has been shown that purified recombinant E . coli derived TNF-a also has cytostatic or cytolytic effects on tumor cell lines (Sugarman et al., 1985; Wang et al., 1985) and tumor 1 Abbreviations: TNF-a, tumor necrosis factor a; CD, circular dichroism; DTNB, 5,5’-dithiobis(2-nitrobenzoicacid); DTT, dithiothreitol; SDS, sodium dodecyl sulfate; SDS-PAGE, SDS-polyacrylamide gel electrophoresis; Tris-HC1, tris(hydroxymethy1)aminomethane hydrochloride.
0006-2960/87/0426-1322$01 SO10 0 1987 American Chemical Society
TUMOR NECROSIS FACTOR
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necrosis activity in vivo (Shirai et al., 1985). This has led to clinical investigations of this protein in cancer patients. However, very little has been reported on the structural features of the protein. We have expressed the protein in E . coli and purified it to near homogeneity for structural studies. In this paper, we report results of structure analyses of the purified protein by sulfhydryl titration, gel filtration, and circular dichroism. MATERIALS AND METHODS Materials. Ultrapure urea was obtained from Schwarzl Mann. Cloning and Expression of TNF-a. A synthetic gene coding for human TNF-a was subcloned into an expression vector with a regulatable promoter. A derivative of E . coli strain C600 harboring the TNF-a expression vector was grown and induced to high-level expression by use of standard fermentation processes. TNF-a Preparation. E. coli cells containing TNF-a were broken with a Gaulin homogenizer. After centrifugation of the disrupted cell suspension, the proteins in the pellet and supernatant fractions were analyzed by SDS-PAGE in reducing gels (data not shown). The results showed TNF-a in the supernatant and pellet fractions. The purity was greater in the pellet fraction, since the majority of E . coli derived proteins remain in the supernatant. This provided some advantage for the pellet fraction for purification of TNF-a. In this study, TNF-a was purified from both fractions. The protein was purified by a series of chromatographic procedures, i.e., a cation-exchange chromatography at pH 4.5 followed by an anion-exchange chromatography at pH 9.0. The protein from the pellet fraction was solubilized in concentrated urea in the presence of dithiothreitol (DTT) and purified in the presence of urea and DTT except for the last chromatography, in which DTT, but not urea, was removed to allow oxidation of the sulfhydryl groups in the molecule. This purified protein was diluted into 10 volumes of 40 mM Tris-HC1, pH 8.5, to decrease urea concentration, and concentrated to -2 mg/mL. The concentrated solution was loaded on a Sephadex G-75 column equilibrated with 40 mM Tris-HC1 and 0.1 M NaC1, pH 8.5. The elution pattern showed a small peak at the void volume containing both high-molecular-weight contaminants and aggregates of TNF-a, presumably arising from incorrect folding of the protein. In addition, the elution profile showed a single large peak which was pooled as a final product for characterization. There was no other material detected even at M , 17000. TNF-a was also purified from the supernatant fraction of the ruptured cells. In this purification, no denaturants, detergents, or reducing agents were used. In addition, the protein was never exposed to extreme pH or temperature, to minimize possibilities of conformational changes during the process. Therefore, the protein conformation should be maintained as obtained in the E . coli cells through the purification process, although the cysteine residues are probably reduced initially (Freedman & Millson, 1980). Finally, the purified protein was loaded on the Sephadex G-75 column and showed a small peak at the void volume, which contained high-molecularweight contaminating proteins but no TNF-a. This absence of TNF-a aggregates suggests that the TNF-a aggregates observed in material purified from the pellet fraction are due to incorrect folding. Fractions from the second peak at the included volume were pooled. Spectroscopy. UV absorbance spectra were determined on a Hewlett-Packard Model 845 1A diode spectrophotometer at room temperature. Circular dichroism spectra were deter-
VOL. 26, NO. 5 , 1987
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mined at room temperature on a Jasco Model J-5OOC spectropolarimeter equipped with an Oki If 800 Model 30 computer. The data were expressed as the mean residue ellipticity [e], calculated by using the mean residue weight of 111 for TNF-a. Sulfhydryl Titration. Titration of sulfhydryl groups was carried out with 5,5’-dithiobis(2-nitrobenzoic acid) (DTNB) essentially according to Habeeb (1972). The protein at 1-2 mg/mL was titrated with DTNB in 0.1 M phosphate, pH 8, 0.05% ethylenediaminetetraacetic acid, and 4 M guanidine hydrochloride. As a control, the protein was reduced with DTT, precipitated, and washed with trichloroacetic acid to remove free DTT. The pellet was solubilized with the above solvent and titrated with DTNB. Protein ConcentrationDetermination. Protein concentration was spectrophotometrically determined by using the extinction coefficient at 280 nm of 1.62 for 0.1% TNF-a solution. The extinction coefficient for TNF-a was determined by measuring the optical density of a TNF-a solution in 10 mM ammonium bicarbonate (pH 8.2) and quantitating the amount of the protein in solution by amino acid composition analysis. It was assumed that the extinction coefficient is the same in 10 mM ammonium bicarbonate, pH 8.2, and in the solvents used in this study. Miscellaneous. Gel filtration was carried out on a Sephadex G-75 column (1 X 110 cm) equilibrated with 40 mM TrisHC1, pH 8.5, and 0.1 M NaCl. SDS-PAGE was carried out with the system of Laemmli (1970) using a 15% polyacrylamide gel. RESULTSAND DISCUSSION When a protein is expressed at a high level in E . coli, it is often produced in inclusion bodies that appear in an insoluble pellet after disruption of the cells. Therefore, purification of the protein requires solubilization by a denaturant, such as urea or guanidine hydrochloride, detergents, or extreme pH. In addition, when the protein contains cysteine residues, the protein in the insoluble pellet usually is reduced (Schoemaker et al., 1985). This was also true of TNF-a, since it generated a variety of oligomers, as demonstrated by SDS-PAGE under nonreducing conditions, when an attempt was made to purify the protein by the same procedures as described but without the inclusion of DTT throughout the purification processes. Therefore, if the cysteine residues are in a disulfide bond in the native state, they would have to be oxidized during or after purification. These two factors could result in generation of incorrectly folded molecules during purification. Thus, it would be of interest to compare the protein obtained from the insoluble fraction with that obtained from the soluble fraction. For this purpose, the soluble fraction of TNF-a was purified without using any denaturants and reducing agents so that the protein conformation should be maintained as originally formed in the E . coli cells. Figure 1 shows the SDS-PAGE profile of the purified protein under reducing conditions. TNF-a purified from the insoluble fraction (designated as P-TNF-a) showed essentially a single band at M , 17 000. TNF-a purified from the soluble fraction (designated as S-TNF-a) showed a major band corresponding to TNF-a and a few high-molecular-weight and one low-molecular-weight contaminant band. The densitometric scan indicated that the S-TNF-a preparation is 95% pure. The P-TNF-a and S-TNF-a preparations also showed a major band in nonreducing gel at the same molecular weight as that obtained under reducing conditions. This suggests either that these TNF-a preparations remain reduced when purified or that the reduced and oxidized TNF-a have the
1324 BIOCHEMISTRY
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n o m 2: Gel fillration profile of P-TNF-a and STNF-a in Sephadex G-75 column: broken line, S-TNF-a: solid line, P-TNF-a. FIGURE I: SDEPAGE profile of P-TNF-a and STNF-a: preparations: lana 1 and 3. STNF-a: lanes 2 and 4. P-TNF-a: lanes I and 2. reduced; lanes 3 and 4. nonrcduced
same mobility in SDS-PAGE. However, the former possibility was excluded, as described later. In addition to the major TNF-a band, the P-TNF-a and S-TNF-a preparations showed a dimer band constituting approximately 8% and I I%, respectively, under nonreducing conditions. It appears that the dimer of P-TNF-a has dimerent mobility in SDS-PAGE from that of S-TNF-a, suggesting that they have different intermolecular disulfide bonds. The presence of disulfide-linked dimer in the S-TNF-a preparation suggests that the soluble TNF-u is still reduced in E. coli cells. as shown by Freedman and Hillson (1980). and is oxidized after cell disruption. resulting in partial formation of intermolecular disulfide bonds. The P-TNF-o and S-TNF-u preparations were titrated with DTNB to clarify the state of the cysteine residues. The results showed no reaction with DTNB for both samples in 4 M guanidine hydrochloride in which all the cysteine residues should be fully accessible, indicating no free sulfhydryl groups. When reduced by DTT. P-TNF-a showed reaction with DTNB, and the amount of free sulfhydryl groups calculated was approximately 2 mol/mol of protein. This value quantitatively agrees with the number of cysteine residues in the TNF-o molecule (M,17OOO). Since the reduction wascarried out in 40 mM Tris and 0.1 M NaCI. pH 8.5. without denaturant, the above results suggest that the disulfide bond is at the surface of the protein. Secondary structure prediction according to Chou and Fasman (1978) showed an overall @-turn potential calculated for the sequence of Cys-69 to Thr-72 to be 4.43 X l ( r and that of Ser-99 to Gln-I02 to be 4.14 X IO4. T h e values are about 8-fold higher than the average value obtained by Chou and Fasman for the 8-turn potential of tetrapeptides. Thus, it is very likely that Cys-69 and Cys-101 are at the first and third positions of &turns, respectively. Therefore, they should locate on the protein surface (Kuntz. 1972). consistent with their observed accessibility to DTT. Comparison of the results of sulfhydryl titration and SDS-PAGE indicates that the 17000 band observed under nonreducing conditions has an intramolecular disulfide bond and has the same mobility in SDS-PAGE as that with the disulfide bond reduced. This indicates that the Stokes radius of the SDS/TN F-o complex is not affected by formation of the intramolecular disulfide bond. Gel filtration experiments were performed to estimate the molecular size of TNF-a. Figure 2 shows the elution profiles of S-TNF-a and P-TNF-o with elution positions of standard proteins. Both TNF-a preparations eluted at an identical
elution position. This elution position was essentially identical with that for ovalbumin (M, 46000), which is intermediate in size between the expected dimer and trimer (34000 and 51 OOO,respectively). This molecular weight is identical, within experimental error, with that reported by Aggarwal et al. (1985) for protein purified from a cell line that naturally secretes TNF-a. This result suggests that TNF-a self-associates into an oligomer, although it is not excluded that it is an unusually asymmetric monomer. If self-association ofcurs. then the fact that TNF-a has an intramolecular disulfide bond indicates that the observed self-association occurs by means other than disulfide bonds. CD spatra of P-TNF-a and S-TNF-a are shown in Figure 3. There are no significant differences between them, indicating that P-TNF-a is refolded into a structure apparently identical with that of S-7°F-a. This suggests that TNF-a undergoes no irreversible changes in the presence of concentrated urea and D’IT and can be readily refolded. Reflecting the structural similarity, the cytolytic activities of P-TNF-a and S-TNF-a were identical, within experimental error, when assayed on mouse L929 cells according to the procedure of Spofford et al. (1974). The observed value of 1 X IO’ units/mg is close to the previously reported values (Shirai et al.. 1985: Sugarman et al., 1985). Since the conformations of S-TNF-a and P-TNF-u were identical, the characterization was primarily carried out with the P-TNF-a preparation, which has greater purity. Both P-TNF-a and S-TNF-a were reduced with 20 mM DTT in 40 mM Tris. pH 8.5, and 0.1 M NaCl and dialyzed against 1 mM DTT in the same buffer to lower the DTT concentration for C D measurements. Reduction of the disulfide bond was demonstrated by sulfhydryl titration, which showed an incorporation of about 2.0 mol of DTNBJmole of protein. The C D spectra were essentially identical, within experimental error, with those before reduction in both nearand far-UV regions, suggesting that the disulfide bond plays no detectable role in maintaining the protein conformation at r w m temperature. In addition, it is likely that the disulfide makes no contribution to the aromatic C D spectrum. These results are consistent with the notion that the disulfide bond is at the protein surface. The far-UV spectrum shows a minimum at 219 nm (-6.2 X IO1deg.cm’/dmol) and a maximum at 200 nm (8.7 X IO1 deg.cm*/dmol). This spectrum cannot reasonably be fitted to the spectra generated from combinations of Chang’s model spectra (Chang et al., 1978). A comparison with the C D spectra reported for other proteins showed that the far-UV
VOL. 26, NO. 5 , 1987
TUMOR NECROSIS FACTOR I
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--a
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.-
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WAVELENGTH (nm)
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WAVELENGTH (nm)
3: Circular dichroism spectra of P-TNF-a (solid line) and S-TNF-a (broken line) in 40 m M Tris and 0.1 M NaCI, pH 8.5. Far-UV spectrum of P-TNF-a in 0.95% SDS is shown (dotted line). FIGURE
spectrum of TNF-a is similar to that of concanavalin A, which also could not be fitted to the spectra reconstituted by the same procedure. Concanavalin A has a minimum at 222 nm (-6.7 X lo3 degcm2/dmol) and a maximum at 197 nm (1.1 X IO4 degcm2/dmol), similar to those for TNF-a shown in Figure 3. From X-ray data, concanavalin A consists of 2% helix, 51% @-sheet, 14% @-turns,and 38% random coil (Reeke et al., 1975). Proteins containing a large amount of 0-sheet structure are difficult to fit by using models, because of the wide variety in @-sheetCD spectra. For example, variations in the amount of twist are predicted to greatly affect the spectrum (Illangasekare & Woody, 1986). Assignment of former, indifferent, and breaker features to the residues in the TNF-a sequence indicates a periodic distribution of a-helix breakers, according to Chou and Fasman (1978). This suggests that there are only small fragments (corresponding to at most two turns) that could form a-helix, and these would be too unstable to be maintained. The same analysis shows a periodic distribution of @-sheetbreakers. Therefore, it is likely that the intra- or interchain @-sheetis formed by a series of small fragments with high @-sheetpotential. TNF-a has fairly intense CD in the near-UV region that is probably the sum of contributions from a number of chromophores (Strickland, 1974). (The molecule contains two Trp, seven Tyr, and four Phe.) The peak at 291 nm (-116 degcm2/dmol) can be assigned to Trp, probably the 'Lb transition, although the 'La band could also contribute here. The remaining broad intense CD to shorter wavelengths shows peaks at about 280 and 285 nm ( 162 degcm2/dmol). These could be due to a 'La Trp band and/or one or more Tyr bands. CD spectra of TNF-a in 0.95% SDS were determined, since SDS is known to stabilize a-helix and, in the case of nonhelical proteins, induce a-helix formation (Jirgensons, 1976). This experiment should therefore indicate whether the observed nonhelical nature of the TNF-a is mainly due to its primary structure. A sample solution was prepared by adding 20% SDS to a final concentration of 0.95% into a TNF-a solution in 40 mM Tris and 0.1 M NaCl, pH 8.5. The near-UV CD spectra showed nearly complete loss of the CD signal in the N
region of 245-320 nm, as expected from SDS-induced denaturation. The far-UV spectrum in 0.95% SDS is shown in Figure 3. The calculation according to Greenfield and Fasman (1969) gives an a-helical content of 51% in 0.95% SDS, indicating that a-helix formation is strongly enhanced by SDS. Formation of a-helix in nonhelical proteins with high 0-sheet content like TNF-a has been observed for other proteins (Jirgensons, 1976). It is not certain whether a-helix is formed at the expense of &sheet or of unordered structure. Although it has been shown that TNF-a has an unfavorable sequence for formation of a-helix, this result shows that the amino acid sequence is not the sole determinant of the secondary structure of this particular protein and that environmental factors contribute to formation of secondary structure. CONCLUSIONS TNF-a purified from the reduced, insoluble fraction of E . coli was identical in structure with TNF-a from the soluble fraction. This indicates that TNF-a is readily refolded from the fully unfolded state. Purification of the protein is much easier using the insoluble fraction, since a large portion of the E . coli proteins remain in the supernatant upon cell breakage. Therefore, the observed correct refolding of urea-denatured TNF-a shows an advantage in using the insoluble fraction over the soluble fraction of the protein for purification and hence large-scale production. Comparison of sulfhydryl titration, SDS-PAGE, and gel filtration experiments suggests that TNF-a is a noncovalently associating oligomer. A disulfide bond is formed within the subunit at the protein surface. Reduction of the disulfide bond does not significantly alter the conformation of the protein, which shows that the disulfide bond is not essential in maintaining the overall structure of TNF-a. It is of interest, therefore, to see if the disulfide bond plays any role in the biological activity of TNF-a or in the stability of the proteins. Preliminary experiments suggested that the TNF-a analogues with substitutions at the two cysteine residues have comparable cytolytic activity with the natural sequence, indicating that the disulfide bond is not essential for the activity.
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Biochemistry 1987, 26, 1326-1332
ACKNOWLEDGMENTS We thank R. Everett for amino acid composition analysis, E. Toth for in vitro bioassays, Dr. N. Stebbing for critically reading the manuscript, and J. Bennett for typing. REFERENCES Aggarwal, B. B., Kohr, W. J., Hass, P. E., Moffat, B., Spencer, S. A., Henzel, W. J., Bringman, T. S., Nedwin, G. E., Goeddel, D. V., & Harkins, R. N. (1985) J . Biol. Chem. 260, 2345-2354. Carswell, E. A., Old, L. J., Kassel, R. L., Green, S., Fiore, N., & Williamson, B. (1975) Proc. Natl. Acad. Sci. U.S.A. 72, 3666-3670. Chang, C. T., Wu, C.-S. C., & Yang, J. T. (1978) Anal. Biochem. 91, 13-31. Chou, P. Y., & Fasman, G. D. (1978) Annu. Rev. Biochem. 47, 251-276. Freedman, R. B., & Millson, D. A. (1980) in Enzymology of Post- Translational Modifcation of Proteins (Freedman, R. B., & Hawkins, H. C., Eds.) Vol. 1, pp 157-212, Academic, London. Green, S., Dobrjansky, A., Carswell, E. A., Kassel, R. L., Old, L. J., Fiore, N., & Schwartz, M. K. (1976) Proc. Natl. Acad. Sci. U.S.A. 73, 381-385. Greenfield, N., & Fasman, G. (1969) Biochemistry 8, 4108-41 16. Habeeb, A. F. S. A. (1972) Methods Enzymol. 25,457-465. Hammerstrom, J. (1982) Scand. J . Immunol. 15, 311-318. Helson, L., Green, S., Carswell, E., & Old, L. J. (1975) Nature (London) 258, 731-732. Illangasekare, M. P., & Woody, R. W. (1986) Biophys. J . 49, 296a.
Jirgensons, B. (1976) Biochim. Biophys. Acta 434, 58-68. Kuntz, I. D. (1972) J. A m . Chem. SOC.94, 4009-4012. Laemmli, U. K. (1970) Nature (London) 227, 680-685. Marmenout, A., Fransen, L., Tavernier, J., Van Der Heyden, J., Tizard, R., Kawashima, E., Shaw, A., Johnson, M.-J., Semon, D., Muller, R., Ruysshaert, M.-R., Van Vliet, A., & Fiers, W. (1985) Eur. J . Biochem. 152, 515-522. Matthews, N. (198 1) Immunology 44, 135-142. Matthews, N. (1982) Br. J . Cancer 45, 615-617. Palladino, M. A., Kohr, W. J., Aggarwal, B. B., & Goeddel, D. V. (1984) Nature (London) 312, 724-729. Pennica, D., Nedwin, G. E., Hayflick, J. S., Seeburg, P. H., Derynck, R., Pzlladino, M. A., Kohr, W. J., Aggarwal, B. B., & Goeddel, D. V. (1985) Nature (London) 312, 724-729. Reeke, G. N., Becker, J. W., & Edelman, G. M. (1975) J . Biol. Chem. 250, 1525-1547. Ruff, M. R., & Gifford, G. E. (1981) Infect. Immun. 31, 380-385. Schoemaker, J. M., Brasnett, A. H., & Marston, F. A. 0. (1985) EMBO J . 4, 755-780. Shirai, T., Yamaguchu, H., Ito, H., Todd, C. W., & Wallace, R. B. (1985) Nature (London) 313, 803-806. Spofford, B., Dayness, R. A., & Granger, G. A. (1974) J. Immunol. 112, 2111-2115. Strickland, E. H. (1974) CRCCrit. Rev. Biochem. 2, 113-175. Sugarman, B. J., Aggarwal, B. B., Hass, P. E., Figari, I. S., Palladino, M. A,, Jr., & Shepard, H. M. (1985) Science (Washington, D.C.)230, 943-945. Wang, A. M., Creasey, A. A., Ladner, M. B., Lin, L. S., Strickler, J., Van Arsdell, J. N., Yamamoto, R., & Mark, D. F. (1985) Science (Washington, D.C.) 228, 149-154.
Sequence Comparisons of Complementary DNAs Encoding Aequorin Isotypes Douglas C. Prasher,t Richard 0. McCann,* Mathew Longiaru,* and Milton J. Cormier*s* Department of Biochemistry, University of Georgia, Athens, Georgia 30602, and Hoffmann- La Roche, Nutley, New Jersey 07110 Received September 5, 1986; Revised Manuscript Received November 14, 1986 Aequorin is the Ca2+-activated photoprotein which participates in the bioluminescence from the circumoral ring of the hydromedusa Aequorea victoria. The nucleotide sequences of five aequorin cDNAs have been compared and shown to code for three aequorin isoforms. The cDNA AEQl contains the entire protein coding region of 196 amino acids. The other four cDNAs contain only 70-90% of the coding region and apparently code for at least two other isoforms whose amino acid sequences differ significantly from that encoded by AEQ1. The nucleotide sequences coding for the three isotypes differ at a minimum of 54 positions out of a total of 588 nucleotides necessary to code for apoaequorin. Of these nucleotide differences, 24 account for 23 amino acid replacements, substantiating the microheterogeneity observed during sequencing of purified native aequorin [Charbonneau, H., Walsh, K. A., McCann, R. O., Prendergast, F. G., Cormier, M. J., & Vanaman, T. C. (1985) Biochemistry 24,6762-67711. Comparison of the deduced c D N A translations with the native protein sequences suggests the loss of seven residues from the amino terminus during purification of aequorin from Aequorea. Aequorin rapidly extracted from the jellyfish using conditions to minimize proteolysis is shown to have a larger molecular weight than that of purified native aequorin. Escherichia coli expressed aequorin encoded by AEQl is shown to have the same molecular weight and isoelectric point as those of one of the isotypes rapidly extracted from Aequorea. ABSTRACT:
O r g a n i s m s within each of four kingdoms are bioluminescent; they include bacteria, animals (insects, fish, earthworms), *Address correspondence to this author. *University of Georgia. Hoffmann-La Roche.
*
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protoctists (dinoflagellates), and fungi (Herring, 1978). A majority of bioluminescent animals are of marine origin and include a large number of coelenterates (cnidarians and ctenophores). These coelenterate luminescent species have closely related bioluminescent systems (Hori et al., 1973, 1977; Ward & Cormier, 1975). Those systems found in the cni-
0 1987 American Chemical Society
