Overexpression in Escherichia coli of chemically synthesized gene for active 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 oligodeoxynucleotides which were chemically synthesized. The synthetic gene was introduced into vector pET15b for expression in Escherichia coli BL21(DE3) under the control of T7 promoter. However, in SDS-PAGE and Western blotting analyses of the E. coli cell lysate, the expression product could not be detected. Expression analysis for various partially deleted gene fragments suggested that the putative hairpin-like structure of mRNA in the 5'-terminal coding region might interrupt efficient expression. When the hairpin structure was eliminated by using degenerate codons, the resulting gene could be overexpressed in E. coli. Although the gene product was accumulated in an insoluble fraction as inclusion bodies, its inhibitory activity could be recovered by solubilization with 8 M urea, followed by refolding through two successive steps of dialysis at alkaline pH. After purification, the recombinant 0.19 alpha-AI showed the same characteristics as the authentic inhibitor in terms of N-terminal amino acid sequence, peptide mapping on reverse-phase HPLC, far-UV circular dichroism (CD) spectrum and have inhibition of human salivary alpha-amylase. Thus, we have established an overexpression system in E. coli for active recombinant 0.19 alpha-AI.
Okuda
M
M
Department of Chemistry, College of Science and Engineering, Aoyama Gakuin University, Tokyo.
Satoh
T
T
Sakurai
N
N
Shibuya
K
K
Kaji
H
H
Samejima
T
T
D000595:Amino Acid Sequence; D000681:Amylases / Q000037:antagonists & inhibitors; D001483:Base Sequence; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics*; D015964:Gene Expression Regulation, Bacterial; D017343:Genes, Plant; D005813:Genes, Synthetic; D005822:Genetic Vectors / Q000138:chemical synthesis*; D008969:Molecular Sequence Data; D010940:Plant Proteins / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D012639:Seeds / Q000737:chemistry / Q000235:genetics; D014908:Triticum / Q000737:chemistry / Q000235:genetics*; D014361:Trypsin Inhibitors / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification; D000516:alpha-Amylases / Q000037:antagonists & inhibitors
D010940:Plant Proteins; D011994:Recombinant Proteins; D014361:Trypsin Inhibitors; D000681:Amylases; D000516:alpha-Amylases