CLONING AND STUDIES OF NEUROSPORA ALKALINE PROTEASE

THE DEVELOPMENT OF MICROBIAL SYSTEMS FOR EXPRESSION OF HETEROLOGOUS PROTEINS HAS ENABLED THE ISOLATION OF GREATER AMOUNTS OF SOURCE PROTEINS, OFTEN IN GREATER PURITY THAN IS POSIBLE FROM THE ORIGINAL SOURCE. PROTEINS SO EXPRESSED HAVE APPLICATIONS RANGING FROM INDUSTRIAL USE, THROUGH PROTEIN BIOCHEMISTRY RESEARCH, TO HUMAN THERAPEUTICS. RESEARCHERS AT HAWAII BIOTECHNOLOGY GROUP HAVE DEMONSTRATED HETEROLOGOUS PROTEIN EXPRESSION IN NEUROSPORA CRASSA USING TWO PROMOTERS-THE GLUCOSE REPRESSIBLE PROMOTER OF GRG-L AND THE CONSTITUTIVE PROMOTER OF THE BETA-TUBULIN GENE. NEUROSPORA TRANSFORMANTS EXPRESSING A BOVINE PREPROCHYMOSIN CDNA ACCUMULATE SOLUBLE, ENZYMATICALLY ACTIVE MATURE CHYMOSIN IN THE CULTURE MEDIUM. ONE IMPORTANT ELEMENT OF HETEROLOGOUS PROTEIN EXPRESSION SYSTEMS IS THE APPROPRIATE HOST STRAIN, FOR WHICH PROTEASE-DEFICIENT MUTANTS ARE OFTEN SUPERIOR. NEUROSPORA SECRETES AN ALKALINE PROTEASE IN THE PRESENCE OF PROTEIN IN THE CULTURE MEDIUM WHEN ALSO STARVED FOR CARBON, NITROGEN, OR SULFUR. WHEN INDUCED, THE ALKALINE PROTEASE COMPRISES 40 PERCENT OF SECRETED PROTEINS. TO DEVELOP BOTH AN IMPROVED HOST AND ADDITIONAL EXPRESSION VECTORS FOR EXPRESSION OF HETEROLOGOUS PROTEINS, RESEARCHERS ARE CLONING CDNA AND THE GENOMIC LOCUS ENCODING THE N. CRASSA SECRETED ALKALINE PROTEASE. THESE CLONES WILL BE USED TO CREATE ALKALINE PROTEASE-DEFICIENT DISRUPTION MUTATIONS AND TO ADD THE PROTEASE PROMOTER AND SECRETION SIGNAL PEPTIDE TO THE EXPRESSION VECTOR REPERTOIRE.