title: Expression in Escherichia coli of active human alcohol dehydrogenase lacking N-terminal acetylation.
abstract: Human alcohol dehydrogenase (ADH, beta beta 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 beta-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% 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 beta-subunit except that the N-terminus was non-acetylated, indicating a correct removal of the initiator methionine, but lack of further processing.
authors.affiliation: Department of Chemistry 1, Karolinska Institutet, Stockholm, Sweden.
authors.forename: J O
authors.initials: JO
authors.lastname: Höög
authors.forename: M
authors.initials: M
authors.lastname: Weis
authors.forename: M
authors.initials: M
authors.lastname: Zeppezauer
authors.forename: H
authors.initials: H
authors.lastname: Jörnvall
authors.forename: H
authors.initials: H
authors.lastname: von Bahr-Lindström
mesh_terms: D000107:Acetylation; D000426:Alcohol Dehydrogenase / Q000032:analysis / Q000302:isolation & purification / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics*; D005822:Genetic Vectors; D006801:Humans; D010957:Plasmids
chemical_list: D000426:Alcohol Dehydrogenase