Recombinant human insulin - VIII. Isolation of fusion protein-S-sulfonate,biotechnological precursor of human insulin, from the biomass of transformed Escherichia coli cells

Various methods have been investigated for the isolation and purification o f fusion proteins of precursors of human insulin in the form of S-sulfonate s, from the biomass of transformed Escherichia coli cells, Fusion proteins were prepared with different sizes and structures of the leader peptide and the poly-His position (inserted for purification by metal chelate affinity chromatography), The fusion proteins contained an IgG binding B domain of protein A from Staphylococcus aureus at the N-terminus and an Arg residue b etween the leader peptide of the molecule and the proinsulin sequence, for trypsin cleavage of the leader peptide. Six residues of Cys in proinsulin a llow the chemical modification of the protein as a (Cys-S-SO3-)(6) derivati ve (S-sulfonate), which increases its polyelectrolytic properties and impro ves the efficiency of its isolation, Various methods of oxidative sulfitoly sis were compared with catalysis by sodium tetrathionate or cystine and Cu2 + or Ni2+ ions. An optimum scheme for the isolation and purification of S-s ulfonated fusion proteins was developed by the combination of metal-chelati ng affinity and ion-exchange chromatography, Highly purified (95%) S-sulfon ated fusion protein was recovered which was 85% of the fusion protein conta ined in the biomass off. coli cells. Folding of fusion protein S-sulfonate occurred with high yield (up to 90-95%). We found that the fusion protein-S -sulfonate has proinsulin-like secondary structure. This structure causes h ighly efficient fusion protein folding. (C) 2001 Academic Press.