Novel pro-insulin having specific amino acid and/or nucleic acid modifications suitable for improved methods of insulin production are provided. Novel and highly efficient processes for preparing the pro-insulin preparations and preparations containing them are also disclosed. The novel pro-insulin preparations may be converted into human insulin useful in therapeutic preparations. Novel peptides of the C-peptide, and N terminus, including RREAEALQVGQVELGGGPGAGSLQPLALEGSLQAR (SEQ ID NO: 32), and MHHHHHHGGR (SEQ ID NO: 2) respectively are provided, as well as the unique nucleic acid molecules encoding them.