Expression in Escherichia coli of active human alcohol dehydrogenase lacking N-terminal acetylation.
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.
Höög
J O
JO
Department of Chemistry 1, Karolinska Institutet, Stockholm, Sweden.
Weis
M
M
Zeppezauer
M
M
Jörnvall
H
H
von Bahr-Lindström
H
H
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
D000426:Alcohol Dehydrogenase