PROCESSING OF MUTATED PROINSULIN WITH TETRABASIC CLEAVAGE SITES TO BIOACTIVE INSULIN IN THE NONENDOCRINE CELL-LINE, COS-7

The amino acid sequence, Arg-4-X-3-Lys/Arg-2-Arg-1 down X+1, is thought to be a concensus processing site for a constitutive secretory pathway in non-endocrine cells. We created a mutant proinsulin DNA with a peptide structure of B chain-Arg-Arg-Lys-Arg-C peptide-Arg-Arg-Lys-Arg-A chain, which compares to the native proinsulin structure of B chain-Arg-Arg-C peptide-Lys-Arg-A chain. When the mutant insulin was expressed in a monkey kidney-derived cell line, COS-7, approximately 60% of the total immunoreactive insulin appeared as mature insulin in the culture medium. This conversion to the mature form was strikingly facilitated by co-expressing the mutant proinsulin with furin, a homologue of the yeast endoprotease, Kex2.