METAL AFFINITY ENGINEERING OF PROINSULIN CARRYING GENETICALLY ATTACHED (HIS)(10)-X-MET AFFINITY TAIL AND REMOVAL OF THE TAG BY CYANOGEN-BROMIDE

An E. coli expression clone coding for human proinsulin, which was fus ed to NH2-terminal beta-galactosidase, was engineered for the separati on from host proteins by introducing peptide devices, and for the sequ ential removal of the fused polypeptide by cyanogen bromide in front o f the NH2 terminal residue (methionine) of the human proinsulin gene. Short synthetic genes encoding oligopeptide residues including (Glu)(n ), (His)(n), (Trp)(n), and (Ser)(n) (n = 10 or 11), which have certain characteristic physical properties such as metal-affinity, polarity, hydrophobicity, and hydrophilicity, respectively, were inserted at the junction region of the gene fusion. Interestingly, it was found that among the oligopeptides, the oligohistidine residue as an affinity-tag has greatly facilitated the procedures for FPI purification, particul arly in the manner of selective metal-affinity precipitation. The chel ating peptide covering the NH2-terminal beta-galactosidase portion cou ld then be removed simply after purification to generate a protein wit h the natural amino acid sequence of proinsulin by cyanogen bromide.