Overexpression of plant histidinol dehydrogenase using a baculovirus expression vector system.
A cDNA encoding cabbage histidinol dehydrogenase, including the chloroplast transit peptide sequence, was overexpressed using a baculovirus expression vector system. The maximum level of the expression of histidinol dehydrogenase was reached 5 days after infection of the insect cells. Two forms of recombinant histidinol dehydrogenase with molecular masses of 53 and 52 kDa, respectively, were obtained by a one-step purification from the cell homogenate. Compared with the 52-kDa form, the 53-kDa form contained 10 additional amino acids at the N-terminus derived from the transit peptide. By incubating the cell homogenate for 2 h at 30 degrees C, the 53-kDa form could be completely converted to the 52-kDa form. This conversion was blocked by leupeptin. Eighty percent of the converted 52-kDa form had Cys at position 31 at the N-terminal amino acid and the rest had Met 33. Kinetic properties of the recombinant enzyme were virtually identical to those of histidinol dehydrogenase isolated from cabbage plants. The overexpression of recombinant cabbage histidinol dehydrogenase in insect cells, the proteolytic processing of the preprotein next to the N-terminus (compared to the mature cabbage enzyme), and its easy purification allow the preparation of large amounts of the active enzyme for structural and functional studies.
Nagai
A
A
International Research Laboratories, Ciba-Geigy (Japan) Ltd., Takarazuka.
Suzuki
K
K
Ward
E
E
Moyer
M
M
Hashimoto
M
M
Mano
J
J
Ohta
D
D
Scheidegger
A
A
D000429:Alcohol Oxidoreductases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D016367:Baculoviridae / Q000235:genetics*; D001937:Brassica / Q000201:enzymology*; D002478:Cells, Cultured; D003001:Cloning, Molecular; D004247:DNA / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D005822:Genetic Vectors; D007700:Kinetics; D008969:Molecular Sequence Data; D009036:Moths / Q000166:cytology; D011499:Protein Processing, Post-Translational; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism
D011994:Recombinant Proteins; D004247:DNA; D000429:Alcohol Oxidoreductases; C021358:histidinol dehydrogenase