Molecular cloning of a fungal cDNA encoding protein disulfide isomerase.
Based on the partial amino acid sequences of a protein disulfide isomerase (PDI) from Humicola insolens, two primers were synthesized for reverse transcriptase mediated polymerase chain reaction (RT-PCR) of a fungal RNA. A 0.2-kbp fragment around the consensus sequence of PDIs was obtained and used as a probe for screening a fungal cDNA library. A cDNA clone of PDI from H. insolens was isolated and encoded a polypeptide consisting of 505 amino acids, which was characterized by a N-terminal signal sequence composed of 20 amino acids, a consensus sequence (WCGHCK) at two positions, and a C-terminal endoplasmic reticulum retention signal (HDEL). Bacillus brevis harboring an expression plasmid bearing the fungal PDI cDNA was prepared and its culture supernatant showed a significant PDI activity. This indicates that glycosylation of a fungal PDI is not essential for the enzymatic activity related to an interchange of disulfide bonds.
Kajino
T
T
Toyota Central Research and Development Laboratories Inc., Aichi, Japan.
Sarai
K
K
Imaeda
T
T
Idekoba
C
C
Asami
O
O
Yamada
Y
Y
Hirai
M
M
Udaka
S
S
D000595:Amino Acid Sequence; D001407:Bacillus / Q000235:genetics / Q000378:metabolism; D001483:Base Sequence; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000737:chemistry; D004271:DNA, Fungal / Q000737:chemistry / Q000235:genetics*; D004591:Electrophoresis, Polyacrylamide Gel; D007535:Isomerases / Q000737:chemistry / Q000235:genetics*; D003904:Mitosporic Fungi / Q000201:enzymology / Q000235:genetics*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010957:Plasmids; D016133:Polymerase Chain Reaction; D019704:Protein Disulfide-Isomerases; D017386:Sequence Homology, Amino Acid
D018076:DNA, Complementary; D004271:DNA, Fungal; D007535:Isomerases; D019704:Protein Disulfide-Isomerases