Expression inEscherichia coli of active human alcohol dehydrogenase lacking N-terminal acetylation |
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Authors: | Jan-Olov Höög Marianne Weis Michael Zeppezauer Hans Jörnvall Hedvig von Bahr-Lindstrom |
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Affiliation: | (1) Department of Chemistry I, Karolinska Institutet, S-104 01 Stockholm, Sweden;(2) Department of Biochemistry, Universität des Saarlandes, D-6600 Saarbrücken, FRG |
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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. |
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Keywords: | alcohol dehydrogenase expression plasmid immunodetection N-terminal processing E. coli |
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