Construction of an Expression System for Aqualysin I in Escherichia coli That Gives a Markedly Improved Yield of the Enzyme Protein
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概要
- 論文の詳細を見る
An expression system for aqualysin I from Thermus aquaticus YT-1, a thermophilic serine protease belonging to the proteinase K family, in Escherichia coli is available, but the efficiency of production has been rather low for detailed analysis of the product. We developed a maltose biding protein (MBP)-fused proaqualysin I expression plasmid (pMAQ-c2Δ) in which MBP is attached to the N-terminus of proaqualysin I. MBP appeared effectively to suppress the folding-promoting activity of the N-terminal propeptide when the bacteria were grown at 30 °C, leading to a massive accumulation of fusion aqualysin I precursor. The precursor was converted efficiently to mature aqualysin I by heat treatment at 70 °C, enabling us to obtain 40 times more aqualysin I than is available using expression systems such as pAQNΔC105. By analyzing the product of the pMAQ-c2Δ-derived inactive mutant expression vector, pMAQ-S222A, it was confirmed that aqualysin I was initially expressed as a whole fusion protein and then processed autocatalytically.
- 社団法人 日本農芸化学会の論文
著者
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TOKI Tsutomu
Department of Pediatrics, Hirosaki University School of Medicine
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SAKAGUCHI Masayoshi
Department of Medicine, Shiga University of Medical Science
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KAWAKITA Masao
Department of Physiological Chemistry, The Tokyo Metropolitan Institute of Medical Science
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TAKEZAWA Makoto
Department of Applied Chemistry, Kogakuin University
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SUGAHARA Yasusato
Department of Applied Chemistry, Kogakuin University
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NIIMIYA Keisuke
Department of Applied Chemistry, Kogakuin University
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