Sequencing and high expression of aminopeptidase P gene from Escherichia coli HB101.
A plasmid pAPP1 with a 4 kbp insert at the PstI site of pBR322, encoding aminopeptidase P gene of Escherichia coli HB101 (Yoshimoto et al. (1988) J. Biochem. 104, 730-734), was subcloned into pUC18 and pUC19. The transformant of E. coli JM83 harboring pAPP4 with a 1.9 kbp fragment showed more than 50-fold higher enzyme activity than that of the host, after cultivation at 37 degrees C for 40 h in LB-medium containing ampicillin. When the gene DNA was inserted reversely in pAPP4, the enzyme productivity decreased markedly. The whole nucleotide sequence of the inserted fragment of plasmid pAPP4 was clarified by the dideoxy chain-terminating method. Within this sequence, the mature enzyme protein-encoding sequence was found to start just after an ATG codon, as judged by comparison with amino-terminal protein sequencing. Eleven bases upstream from the proposed initiation codon was an AGGAGA sequence which seemed to be a ribosome binding site. Thirty-four bases upstream from the proposed start codon was the 6-base sequence TACAAA, the so-called -10 region or Pribnow box. Further, the 6-base sequence TTTACT around 77 bases upstream from the start codon was deduced to be a putative -35 region consensus sequence. The inverted repeat at 1334 was tentatively assumed to be a terminator. The molecular weight of the enzyme was estimated to be 49,650 from the nucleotide sequence. The purified enzyme contained 0.2 gram atom of zinc per subunit. The enzyme activity was inhibited by EDTA and activated 5-fold by Mn2+.
Yoshimoto
T
T
School of Pharmaceutical Sciences, Nagasaki University.
Tone
H
H
Honda
T
T
Osatomi
K
K
Kobayashi
R
R
Tsuru
D
D
D000595:Amino Acid Sequence; D000626:Aminopeptidases / Q000096:biosynthesis / Q000235:genetics*; D001483:Base Sequence; D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000235:genetics*; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics / Q000378:metabolism*; D005786:Gene Expression Regulation; D005798:Genes, Bacterial; D008969:Molecular Sequence Data; D010957:Plasmids; D014169:Transformation, Bacterial
D004269:DNA, Bacterial; D000626:Aminopeptidases; C022750:X-Pro aminopeptidase