Over-expression of Escherichia coli F₁F_o–ATPase subunit a is inhibited by instability of the uncB gene transcript

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• 作者：Arechaga ; Ignacio ; Miroux ; Bruno ; Runswick ; Mike J. ; Walker ; John E.
• 关键词：Membrane protein ; Over-expression ; ATP synthase ; mRNA degradation
• 刊名：FEBS Letters
• 出版年：2003
• 出版时间：July 17, 2003
• 年：2003
• 卷：547
• 期：1-3
• 页码：97-100
• 全文大小：239 K

文摘

Little is known about the stability of transcripts encoding membrane proteins in strong expression systems and its effect on membrane protein over-production. We have expressed all the genes encoding subunits of the membrane domain F_o of the ATP synthase in a T7 RNA polymerase-based system. All of them but uncB (subunit a) were expressed separately at very high levels in the bacterial hosts Escherichia coli C41(DE3) and C43(DE3). However, expression of uncB was extremely toxic to the bacteria. Northern blot analysis showed that the level of accumulation of the mRNA from uncB was very low. Deletion of uncB in combination with gene fusion experiments demonstrated that the middle region of the gene, encoding amino acids 92–171, exhibited a dominant toxic phenotype associated with a very poor level of expression. Green fluorescent protein fusions with N- and C-ends of uncB helped to stabilize the mRNA and to obtain high yields of protein.