Overexpression in Escherichia coli of chemically synthesized gene for active 0.19 α-amylase inhibitor from wheat kernel

Masahiko Okuda, Takanori Satoh, Nobuhiko Sakurai, Kazunori Shibuya, Hiroyuki Kaji, Tatsuya Samejima

Research output: Contribution to journalArticle

9 Citations (Scopus)

Abstract

A synthetic gene encoding 0.19 α-amylase inhibitor (α-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 α-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 α-amylase. Thus, we have established an overexpression system in E. coli for active recombinant 0.19 α-AI.

Original languageEnglish
Pages (from-to)918-926
Number of pages9
JournalJournal of Biochemistry
Volume122
Issue number5
Publication statusPublished - 1997 Nov

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Amylases
Escherichia coli
Triticum
Genes
Synthetic Genes
Gene encoding
Peptide Mapping
Dialysis
Oligodeoxyribonucleotides
Inclusion Bodies
Circular Dichroism
Codon
Purification
Urea
Polyacrylamide Gel Electrophoresis
Amino Acid Sequence
Western Blotting
High Pressure Liquid Chromatography
Amino Acids
Messenger RNA

Keywords

  • α-amylase inhibitor
  • Hairpin structure
  • Recombinant
  • Synthetic gene
  • Wheat kernel

ASJC Scopus subject areas

  • Biochemistry

Cite this

Overexpression in Escherichia coli of chemically synthesized gene for active 0.19 α-amylase inhibitor from wheat kernel. / Okuda, Masahiko; Satoh, Takanori; Sakurai, Nobuhiko; Shibuya, Kazunori; Kaji, Hiroyuki; Samejima, Tatsuya.

In: Journal of Biochemistry, Vol. 122, No. 5, 11.1997, p. 918-926.

Research output: Contribution to journalArticle

Okuda, Masahiko ; Satoh, Takanori ; Sakurai, Nobuhiko ; Shibuya, Kazunori ; Kaji, Hiroyuki ; Samejima, Tatsuya. / Overexpression in Escherichia coli of chemically synthesized gene for active 0.19 α-amylase inhibitor from wheat kernel. In: Journal of Biochemistry. 1997 ; Vol. 122, No. 5. pp. 918-926.
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