Metal Affinity Engineering of Proinsulin Carrying Genetically Attached (His)_<10>-X-Met Affinity Tail and Removal of the Tag by Cyanogen Bromide

概要

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An E. coli expression clone coding for human proinsulin, which was fused to NH_2-terminal β-galactosidase, was engineered for the separation from host proteins by introducing peptide devices, and for the sequential removal of the fused polypeptide by cyanogen bromide in front of the NH_2 terminalresidue (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, particularly in the manner of selective metal-affinity precipitation. The chelating peptide covering the NH_2-terminal beta-galactosidase portion could then be removed simply after purification to generate a protein with the natural amino acid sequence of proinsulin by cyanogen bromide.
社団法人日本農芸化学会の論文
1994-09-23