The latest data on hcterologous gene expression in fungi indicate that fungi express and sccrete significant amounts of heterologous protein under appropriate culture conditions, e.g. (1). This encouraged us to start our research on human insulin production in Aspergillus. The first aim of our work was to synthesize the human proinsulin gene and to clone it in fungal expression vector. Various mutations were introduced in the sequence of human proinsulin gene to study the (hopefully) positive effects of these mutations on protein secretion. The human proinsulin gene was synthesized by PCR based on overlapping, complementary oligonucleotides used as templates and primers at the same time. The synthesis was performed in fragments of approximately 100-200 bp, which were joined together in pUC19 resulting in the following proinsulin genes: 1) wt proinsulin, using codons favored for efficient expression in A. niger; 2) proinsulin with a C-chain carrving a N-glycosylation consensus; 3) proinsulin from which most of the C-chain is deleted and 4) proinsulin from which the complete C-chain is deleted. Different genes were cloned into fungal expression vectors based on either the glaA promoter and signal sequence or the glaA promoter and the entire glucoamylase coding region (as secretion carrier). The various insulin expression vectors were introduced in A. niger and insulin activity was determined in culture supernatants of obtained transformants. (1)M. P. Brockhuijsen et al (1993) J. Blotechnol. 31: 135-145.
Full conference title:
3rd EUROPEAN CONFERENCE ON FUNGAL GENETICS
- ECFG 3rd (1996)