Abstract
Human alcohol dehydrogenase (ADH, tiff isozyme of class I) was expressed in Escherichia coli, purified to homogeneity, and characterized regarding N-terminal processing. The expression system was obtained by ligation of a cDNA fragment corresponding to the fl-subunit of human liver alcohol dehydrogenase into the vector pKK 223-3 containing the tac promoter. The enzyme, detected by Western-blot analysis and ethanol oxidizing activity, constituted up to 3 ~o of the total amount of protein. Recombinant ADH was separated from E. coli ADH by ion-exchange chromatography and the isolated enzyme was essentially pure as judged by SDS-polyacrylamide gel electrophoresis and sequence analysis. The N-terminal sequence was identical to that of the authentic fl-subunit except that the N-terminus was non-acetylated, indicating a correct removal of the initiator methionine, but lack of further processing.
This is a preview of subscription content, log in via an institution to check access.
References
Vallee, B. L. and Bazzone, T. J. (1982). In:Isozymes: Current Topics in Biological and Medical Research (M. C. Rattazzi, J. G. Scandalios and G. S. Witt, Eds.), Vol 8, pp. 219–244.
Jörnvall, H., Höög, J.-O., von Bahr-Lindström, H. and Vallee, B. L. (1987),Proc. Natl. Acad. Sci. USA 84:2580–2584.
Bühler, R., Hempel, J., Kaiser, R., von Wartburg, J.-P., and Vallee, B. L., Jörnvall, H. (1984),Proc. Natl. Acad. Sci. USA 81: 6320–6324.
Smith, M., Hopkinson, D. A. and Harris, H. (1971).Ann. Hum. Genet. 34:251–271.
Jörnvall, H., von Bahr-Lindström, H. and Höög, J.-O (1988) In:Human Metabolism of Alcohol (R. D. Batt and K. Crow, Eds.), CRC, in press.
von Bahr-Lindström, H., Höög, J.-O., Hedén L.-O., Kaiser, R., Fleetwood, L. Larsson, K., Lake, M., Holmquist, B., Holmgren A., Hempel, J., Vallee, B. L. and Jörnvall, H. (1986).Biochemistry 25:2465–2470.
Hedén, L.-O., Höög, J.-O., Larsso, K., Lake, M., Lagerholm, E., Holmgren, A., Vallee, B. L. Jörnvall, H. and von Bahr-Lindström, H. (1986).FEBS Lett.194:327–332.
Gibson, T. J. (1984).Ph.D. Thesis University of Cambridge.
Amann, E., Brosius, J., and Ptashne, M. (1983).Gene 25:167–178.
Kalb, V. F. and Bernlohr, R. W. (1977).Anal. Biochem. 82:362–371.
Wagner, F. W., Burger, A. R. and Vallee, B. L. (1983).Biochemistry 22:1857–1863.
Morrissey J. (1981).Anal. Biochem. 117:307–310.
Kaiser, R., Holmquist, B., Hempel, J., Vallee, B. L. and Jörnvall, H. (1988).Biochemistry in press.
Kozak, M. (1983).Microbiol. Rev. 47:1–45.
Meyhach, B., Pfleiderer, G. and Mennigmann, H. D. (1973).Eur. J. Biochem.,33: 19–29.
Lange, L. G. and Vallee, B. L. (1976).Biochemistry 15:4681–4686.
Hempel, J., Bühler, R., Kaiser, R., Holmquist, B., de Zalenski, C., von Wartburg, J.-P., Vallee, B. L. and Jörnvall, H. (1984).Eur. J. Biochem. 145:437–445.
Flinta, C., Persson, B., Jörnvall, H. and von Heijne, G. (1986).Eur. J. Biochem. 154:193–196.
Ben-Bassat, A., Bauer, K., Chang, S.-Y., Myambo, K., Boosman, A. and Chang, S. (1987).J. Bacteriol. 169:751–757.
Persson, B., Flinta, C., von Heijne, G. and Jörnvall, H. (1985).Eur. J. Biochem. 152:523–527.
Jörnvall, H., Fairwell, T., Kratofil, P. and Wills, C. (1980).FEBS Lett. 111:214–218.
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Höög, JO., Weis, M., Zeppezauer, M. et al. Expression inEscherichia coli of active human alcohol dehydrogenase lacking N-terminal acetylation. Biosci Rep 7, 969–974 (1987). https://doi.org/10.1007/BF01122131
Received:
Issue Date:
DOI: https://doi.org/10.1007/BF01122131