Abstract

I constructed a genomic DNA library of Aspergillus niger , and determined that it contained 50 000 independent clones of an average size of 14 kb. This library contained approximately twenty genome equivalents. I also constructed an expression vector using the plasmid pGEX-2T to express the fusion protein GST:CREA. This construct contained the full coding sequence of the creA gene of A. niger . The GST affinity tag is located as an amino-terminal fusion with respect to CREA. Induction of the fusion protein was performed under various conditions, and it was concluded that a construct containing a carboxy-terminal affinity tag would ameliorate the yield of full length protein, along with the expression of the construct in the Rosetta © strain of cells produced by Novagen. The protein was purified over a Glutathione Sepharose 4B column from Amersham Pharmacia Biotech, and used for electrophoretic mobility shift assays with oligonucleotides containing the CREA consensus binding sequence, and mutated segments of the glaA promoter of A. niger . Shifted bands were observed in the presence of the purified protein, and competition between labeled and unlabeled DNA indicated clearly that the protein was binding to the CREA consensus binding sequence.