Abstract
The mammalian aspartic proteinases procathepsin D and pepsinogen form insoluble inclusion bodies when expressed in bacteria. They become soluble but nonnative when synthesized as fusions to the carboxy terminus of E. coli maltose-binding protein (MBP). Since these nonnative states of the two aspartic proteinases showed no tendency to form insoluble aggregates, their biophysical properties were analyzed. The MBP portions were properly folded as shown by binding to amylose, but the aspartic proteinase moieties failed to bind pepstatin and lacked enzymatic activity, indicating that they were not correctly folded. When treated with proteinase K, only the MBP portion of the fusions was resistant to proteolysis. The fusion between MBP and cathepsin D had increased hydrophobic surface exposure compared to the two unfused partners, as determined by bis-ANS binding. Ultracentrifugal sedimentation analysis of MBP–procathepsin D and MBP–pepsinogen revealed species with very large and heterogeneous sedimentation values. Refolding of the fusions from 8 M urea generated proteins no larger than dimers. Refolded MBP–pepsinogen was proteolytically active, while only a few percent of renatured MBP–procathepsin D was obtained. The results suggest that MBP–aspartic proteinase fusions can provide a source of soluble but nonnative folding states of the mammalian polypeptides in the absence of aggregation.
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REFERENCES
Betton, J. M., and Hofnung, M. (1994). EMBO J. 13, 1226–1234.
Betton, J. M., Martineau, P., Saurin, W., and Hofnung, M. (1993). FEBS Lett. 325, 34–38.
Betts, S., Haase-Pettingell, C., and King, J. (1997). Adv. Protein Chem. 50, 243–264.
Beyer, B. M., and Dunn, B. M. (1996). J. Biol. Chem. 271, 15590–15596.
Bowden, G. A., Paredes, A. M., and Georgiou, G. (1991). Bio/Technology 9, 725–730.
Brems, D. N., Plaisted, S. M., Kauffman, E. W., and Havel, H. A. (1986). Biochemistry 25, 6539–6543.
Conner, G. E. (1989). Biochem. J. 263, 601–604.
Conner, G. E., and Richo, G. (1992). Biochemistry 31, 1142–1147.
Conner, G. E., and Udey, J. A. (1990). DNA Cell Biol. 9, 1–9.
Cottrell, T. J., Harris, L. J., Tanaka, T., and Yada, R. Y. (1995). J. Biol. Chem. 270, 19974–19978.
Delbruck, R., Desel, C., von Figura, K., and Hille-Rehfeld, A. (1994). Eur. J. Cell Biol. 64, 7–14.
Fortenberry, S. C., Schorey, J. S., and Chirgwin, J. M. (1995). J. Cell Sci. 108, 2001–2006.
Hasilik, A. (1992). Experientia 48, 130–151.
Hasilik, A., and Neufeld, E. F. (1980). J. Biol. Chem. 255, 4937–4945.
Helenius, A., Trombetta, E. S., Hebert, D. N., and Simons, J. F. (1997). Trends Cell Biol. 7, 193–200.
Jaenicke, R. (1987). Prog. Biophys. Mol. Biol. 49, 117–237.
Jaenicke, R. (1991). Biochemistry 30, 3147–3161.
Jaenicke, R. (1995). Phil. Trans. R. Soc. Lond. B Biol. Sci. 348, 97–105.
Jaenicke, R., and Seckler, R. (1997). Adv. Protein Chem. 50, 1–60.
Koelsch, G., Metcalf, P., Vetvicka, V., and Fusek, M. (1995). Adv. Exp. Med. Biol. 362, 273–278.
Kuhelj, R., Dolinar, M., Pungercar, J., and Turk, V. (1995). Eur. J. Biochem. 229, 533–539.
Laemmli, U. K. (1970). Nature 227, 680–685.
LaVallie, E. R., DiBlasio, E. A., Kovacic, S., Grant, K. L., Schendel, P. F., and McCoy, J. M. (1993). Bio/Technology 11, 187–193.
Lin, X. L., Wong, R. N., and Tang, J. (1989). J. Biol. Chem. 264, 4482–4489.
Lin, X., Koelsch, G., Loy, J. A., and Tang, J. (1995). Protein Sci. 4, 159–166.
Liu, G. P., Topping, T. B., Cover, W. H., and Randall, L. L. (1988). J. Biol. Chem. 263, 14790–14793.
Liu, G., Topping, T. B., and Randall, L. L. (1989). Proc. Natl. Acad. Sci. USA 86, 9213–9217.
London, J., Skrzynia, C., and Goldberg, M. E. (1974). Eur. J. Biochem. 47, 409–415.
Marciniszyn, J., Jr., Hartsuck, J. A., and Tang, J. (1976). J. Biol. Chem. 251, 7088–7094.
Netzer, W. J., and Hartl, F. U. (1997). Nature 388, 343–349.
Nilsson, B., and Anderson, S. (1991). Annu. Rev. Microbiol. 45, 607–635.
Sachdev, D., and Chirgwin, J. M. (1998a). Protein Express. Purif. 12, 122–132.
Sachdev, D., and Chirgwin, J. M. (1998b). Biochem. Biophys. Res. Commun. 244, 933–937.
Scarborough, P. E., and Dunn, B. M. (1994). Protein Eng. 7, 495–502.
Schlunegger, M. P., Bennett, M. J., and Eisenberg, D. (1997). Adv. Protein Chem. 50, 61–122.
Teschke, C. M., and King, J. (1995). Biochemistry 34, 6815–6826.
van Holde, K. E., and Weischet, W. O. (1978). Biopolymers 17, 1397–1403.
Wetzel, R. (1994). Trends Biotechnol. 12, 193–198.
Zetina, C. R., and Goldberg, M. E. (1980). J. Mol. Biol. 137, 401–414.
Zettlmeissl, G., Rudolph, R., and Jaenicke, R. (1979). Biochemistry 18, 5567–5571.
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Sachdev, D., Chirgwin, J.M. Properties of Soluble Fusions Between Mammalian Aspartic Proteinases and Bacterial Maltose-Binding Protein. J Protein Chem 18, 127–136 (1999). https://doi.org/10.1023/A:1020663903669
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DOI: https://doi.org/10.1023/A:1020663903669