[4] Thermophilic fungal protein disulfide isomerase

For the study the authors have isolated and characterized a thermostable protein disulfide isomerase (PDI) from a thermophilic fungus, Humicola insolens. The cDNA encoding the fungal PDI has been cloned and expressed in Bacillus brevis. PDIs from vertebrates and yeast are relatively heat labile. Even in the case of an algal enzyme that is most stable of the known PDIs, the stability against heat is not enough for industrial use of the enzyme. Hence, there is continuing interest in finding new, stable PDIs. Refolding activity using scrambled ribonuclease (RNase) as a substrate is discussed. The fungal PDI cDNA is expressed in a heterologous protein production system using B. brevis as a host. In the purification process, anion-exchange chromatography, lectin affinity chromatography, and high-performance liquid chromatography are discussed. The chapter also presents the data for purification of PDI from the fungus, Humicola insolens. To amplify the DNA fragment around the N-terminal consensus region of fungal PDI, two oligonucleotides corresponding to the amino acid sequence are synthesized and used as primers for a reverse transcriptase-mediated polymerase chain reaction (RT-PCR).