Molecular cloning of a fungal cDNA encoding protein disulfide isomerase [from Humicola insolens]

Based on the partial amino acid sequences of a protein disulfide isomerase (PDI) from Humicola insolens, two primers were synthesized for reverse transriptase mediated ploymerase chain reaction (RTPCR) or 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 position, 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