Processing of mutated proinsulin with tetrabasic cleavage sites to bioactive insulin in the non‐endocrine cell line, COS‐7

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Abstract
The amino acid sequence, Arg−4‐X−3‐Lys/Arg−2‐Arg−1 ↓ X+1, is thought to be a consensus 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.