Cloning, sequencing, and expression of the dipeptidyl peptidase IV gene from Flavobacterium meningosepticum in Escherichia coli.
The dipeptidyl peptidase IV (DP IV, EC 3.4.14.5) gene from Flavobacterium meningosepticum was cloned by Southern and colony hybridizations using probes amplified by PCR, and expressed in Escherichia coli DH1. E. coli DH1 harboring pFDP-H1, which was a subclone derived from the positive clone pFDP-1, showed 3.5-fold higher activity than F. meningosepticum. Nucleotide sequencing analysis revealed an open reading frame of 2133 bp, coding for a protein of 711 amino acids with a predicted molecular weight of 80,626. The expressed enzyme in E. coli DH1/pFDP-H1 was purified about 345-fold with an activity recovery of 12.3%. The molecular weight of the purified enzyme was estimated to be 75,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and 160,000 by gel filtration, respectively, suggesting a dimeric form of the native enzyme. The deduced amino acid sequence of DP IV was homologous to those of the serine proteases of the "prolyl endopeptidase family." A sequence near the C-terminal region and the putative catalytic triad residues were well conserved among these enzymes.
Kabashima
T
T
School of Pharmaceutical Sciences, Nagasaki University, Japan.
Yoshida
T
T
Ito
K
K
Yoshimoto
T
T
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D017931:DNA Primers / Q000235:genetics; D004269:DNA, Bacterial / Q000235:genetics; D018819:Dipeptidyl Peptidase 4 / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics*; D005417:Flavobacterium / Q000201:enzymology* / Q000235:genetics*; D015870:Gene Expression; D005798:Genes, Bacterial; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D008970:Molecular Weight; D011487:Protein Conformation; D015183:Restriction Mapping; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity
D017931:DNA Primers; D004269:DNA, Bacterial; D018819:Dipeptidyl Peptidase 4