Armengaud J, Jouanneau Y
Laboratorie de Biochimie Microbienne (CNRS URA 1130, alliée à l'INSERM), Département de Biologie Moléculaire et Structurale, Centre d'Etudes Nucléaires de Grenoble, France.
Protein Expr Purif. 1995 Apr;6(2):176-84. doi: 10.1006/prep.1995.1022.
Different gene expression systems were tested with the aim of overproducing the 7Fe ferredoxin (FdII) from Rhodobacter capsulatus in Escherichia coli. Plasmids bearing the Ptac, ParaB, fdxA gene, encoding FdII, under the control of the PlacUV5, P phi 10 promoters, were compared for their efficiency to promote the synthesis of recombinant ferredoxin. Using a P phi 10-based expression system, recombinant ferredoxin was obtained in a soluble apoform and accumulated up to 20% of the total cell protein. The ferredoxin polypeptide purified from such cells exhibited the correct molecular mass and no detectable heterogeneity when analyzed by mass spectrometry. When other expression systems were used, the ferredoxin was synthesized in holoform but in relatively smaller amounts (0.2 mg/liter of culture). Factors such as promoter strength, efficient translation signals, and stability of the recombinant mRNA were shown to have little effect on the amount of 7Fe ferredoxin produced in E. coli. It is inferred that iron-sulfur clusters insertion may be a rate-limiting factor for synthesis of the 7Fe ferredoxin in the enterobacterium.
为了在大肠杆菌中过量表达来自荚膜红细菌的7Fe铁氧化还原蛋白（FdII），对不同的基因表达系统进行了测试。比较了携带在PlacUV5、P phi 10启动子控制下编码FdII的Ptac、ParaB、fdxA基因的质粒促进重组铁氧化还原蛋白合成的效率。使用基于P phi 10的表达系统，获得了可溶性脱辅基形式的重组铁氧化还原蛋白，其积累量高达总细胞蛋白的20%。从这些细胞中纯化的铁氧化还原蛋白多肽在通过质谱分析时显示出正确的分子量且无可检测的异质性。当使用其他表达系统时，铁氧化还原蛋白以全蛋白形式合成，但量相对较少（0.2毫克/升培养物）。启动子强度、有效翻译信号和重组mRNA稳定性等因素对大肠杆菌中产生的7Fe铁氧化还原蛋白的量影响很小。据推测，铁硫簇插入可能是肠杆菌中7Fe铁氧化还原蛋白合成的限速因素。
