Isoforms of dipeptidyl aminopeptidase IV from Pseudomonas sp. WO24: role of the signal sequence and overexpression in Escherichia coli.
The complete nucleotide sequence of dipeptidyl aminopeptidase IV (DAP IV) from Pseudomonas sp. WO24 was determined. Nucleotide sequence analysis revealed an open reading frame of 2238bp, which was assigned to dap4 by N-terminal and internal amino acid sequences previously reported. The predicted amino acid sequence of DAP IV contains a serine protease Gly-X-Ser-X-Gly-Gly consensus motif and displays extensive homology to DAP IVs and the homologous proteins from eukaryotes and bacteria, belonging to the prolyl oligopeptidase family S9. In Pseudomonas sp. WO24, DAP IV is expressed as 82 and 84-kDa isoforms, having two Met, Met-1 and Met-12, in its N-terminal sequence. Met-1 of DAP IV was mutated to Gly and Met-12 was mutated to Ile, and we overexpressed the two mutated genes in Escherichia coli and obtained the recombinant 82 and 84-kDa proteins from the periplasm and the cytoplasm, respectively, suggesting that the 82 and 84-kDa isoforms are derived from the same gene and localize to different compartments in the cell. We developed purification steps for activting a large amount of 84-kDa isoform protein that will be useful for producing protein for crystallographic studies.
Ogasawara
Wataru
W
Department of Bioengineering, Nagaoka University of Technology, 1603-1 Kamitomioka, Nagaoka, Niigata 940-2188, Japan.
Tanaka
Chiaki
C
Suzuki
Masashi
M
Kobayashi
Go
G
Ogawa
Yoshiyuki
Y
Okada
Hirofumi
H
Morikawa
Yasushi
Y
D000595:Amino Acid Sequence; D000818:Animals; D018819:Dipeptidyl Peptidase 4 / Q000096:biosynthesis* / Q000235:genetics* / Q000302:isolation & purification; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D015971:Gene Expression Regulation, Enzymologic; D006801:Humans; D008969:Molecular Sequence Data; D010802:Phylogeny; D020033:Protein Isoforms; D021382:Protein Sorting Signals / Q000502:physiology*; D011549:Pseudomonas / Q000201:enzymology* / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid
D020033:Protein Isoforms; D021382:Protein Sorting Signals; D011994:Recombinant Proteins; D018819:Dipeptidyl Peptidase 4