MOLECULAR-CLONING OF A FUNGAL CDNA-ENCODING PROTEIN DISULFIDE-ISOMERASE

Based on the partial amino acid sequences of a protein disulfide isome rase (PDI) from Humicola insolens, two primers were synthesized for re verse transcriptase mediated ploymerase chain reaction (RT-PCR) of a f ungal RNA. A 0.2-kbp fragment around the consensus sequence of PDIs wa s 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 polypept ide consisting of 505 amino acids, which was characterized by a N-term inal signal sequence composed of 20 amino acids, a consensus sequence (WCGHCK) at two positions, and a C-terminal endoplasmic reticulum rete ntion signal (HDEL). Bacillus brevis harboring an expression plasmid b earing the fungal PDT cDNA was prepared and its culture supernatant sh owed 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.