著作: Okuda Masahiko/[佐藤 高則]/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/[The Journal of Biochemistry]
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種別 | 必須 | 学術論文(審査論文) | |||
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言語 | 必須 | 英語 | |||
招待 | 推奨 | ||||
審査 | 推奨 | Peer Review | |||
カテゴリ | 推奨 | 研究 | |||
共著種別 | 推奨 | ||||
学究種別 | 推奨 | ||||
組織 | 推奨 | ||||
著者 | 必須 | ||||
題名 | 必須 |
(英) Overexpression in Escherichia coli of Chemically Synthesized Gene for Active 0.19 α-Amylase Inhibitor from Wheat Kernel |
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副題 | 任意 | ||||
要約 | 任意 |
(英) 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. (日) 小麦由来0.19α-アミラ-ゼインヒビタ-(α-AI)をコ-ドする遺伝子を18本のオリゴヌクレオチドを用いて合成した.この合成遺伝子を大腸菌において発現させたところ,封入体として発現した.これを,尿素により可溶化後,溶液中の尿素濃度を減少させることにより,α-AIの立体構造の再生を行なった.得られた組換え体α-AIは,小麦由来のものと同等の阻害活性を示し,他の特性も同じであった. |
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キーワード | 推奨 |
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発行所 | 推奨 | 日本生化学会 | |||
誌名 | 必須 |
The Journal of Biochemistry([日本生化学会])
(pISSN: 0021-924X, eISSN: 1756-2651)
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巻 | 必須 | 122 | |||
号 | 必須 | 5 | |||
頁 | 必須 | 918 926 | |||
都市 | 任意 | ||||
年月日 | 必須 | 1997年 5月 1日 | |||
URL | 任意 | ||||
DOI | 任意 | ||||
PMID | 任意 | 9443806 (→Scopusで検索) | |||
CRID | 任意 | ||||
NAID | 任意 | ||||
WOS | 任意 | A1997YG28000005 | |||
Scopus | 任意 | ||||
評価値 | 任意 | ||||
被引用数 | 任意 | ||||
指導教員 | 推奨 | ||||
備考 | 任意 |
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