OVEREXPRESSION IN ESCHERICHIA-COLI OF CHEMICALLY SYNTHESIZED GENE FORACTIVE 0.19-ALPHA-AMYLASE INHIBITOR FROM WHEAT KERNEL

A synthetic gene encoding 0.19 alpha-amylase inhibitor (alpha-AI) from wheat kernel was obtained by enzymatic assembly of 18 oligodeoxynucle otides which were chemically synthesized. The synthetic gene was intro duced into vector pET15b for expression in Escherichia coli BL21(DE3) under the control of T7 promoter. However, in SDS-PAGE and Western blo tting analyses of the E. coli cell lysate, the expression product coul d not be detected, Expression analysis for various partially deleted g ene fragments suggested that the putative hairpin-like structure of mR NA in the 5'-terminal coding region might interrupt efficient expressi on, When the hairpin structure was eliminated by using degenerate codo ns, the resulting gene could be overexpressed in E. coli. Although the gene product was accumulated in an insoluble fraction as inclusion bo dies, its inhibitory activity could be recovered by solubilization wit h 8 M urea, followed by refolding through two successive steps of dial ysis at alkaline pH. After purification, the recombinant 0.19 alpha-AI showed the same characteristics as the authentic inhibitor in terms o f N-terminal amino acid sequence, peptide mapping on reverse-phase HPL C, far-UV circular dichroism (CD) spectrum and have inhibition of huma n salivary alpha-amylase. Thus, we have established an overexpression system in E. coli for active recombinant 0.19 alpha-AI.