Piubelli L, Aliverti A, Bellintani F, Zanetti G
Dipartimento di Fisiologia e Biochimica Generali, Università di Milano, Italy.
Protein Expr Purif. 1995 Jun;6(3):298-304. doi: 10.1006/prep.1995.1039.
Ferredoxin I is the most abundant form of photosynthetic-type ferredoxin present in spinach chloroplasts. A cDNA clone encoding the precursor of spinach ferredoxin I has been engineered to synthesize the mature form of the plant protein in Escherichia coli. Among several different plasmid constructions, the expression system based on phage T7 promoter (vector pET-11d) was found to be the most efficient for spinach ferredoxin overproduction. Upon induction, ferredoxin I accounted for about 2.5% of soluble E. coli protein. A rapid procedure for the purification of the recombinant protein, which yielded at least 1 mg of homogeneous ferredoxin I per gram of cells (fresh wt), was developed. The recombinant protein was found to be identical to ferredoxin I isolated from spinach, both by mass spectrometry analysis and by N-terminal protein sequencing, indicating in vivo removal of the N-terminal methionine. Ferredoxin I was synthesized as the holoprotein, correctly assembled with the [2Fe-2S] cluster as judged by its absorption spectrum, and was fully active in the assay with its physiological partner (ferredoxin-NADP+ reductase). The expression system described here is amenable to the structure-function relationship study of spinach ferredoxin I through site-directed mutagenesis and NMR spectroscopy.
铁氧化还原蛋白I是菠菜叶绿体中存在的最丰富的光合型铁氧化还原蛋白形式。编码菠菜铁氧化还原蛋白I前体的cDNA克隆已被改造，以便在大肠杆菌中合成该植物蛋白的成熟形式。在几种不同的质粒构建体中，发现基于噬菌体T7启动子的表达系统（载体pET-11d）对菠菜铁氧化还原蛋白的过量生产最为有效。诱导后，铁氧化还原蛋白I占大肠杆菌可溶性蛋白的约2.5%。开发了一种快速纯化重组蛋白的方法，每克细胞（鲜重）至少可产生1毫克纯化的铁氧化还原蛋白I。通过质谱分析和N端蛋白质测序发现，重组蛋白与从菠菜中分离的铁氧化还原蛋白I相同，这表明在体内N端甲硫氨酸已被去除。铁氧化还原蛋白I作为全蛋白合成，根据其吸收光谱判断，它与[2Fe-2S]簇正确组装，并在与其生理伴侣（铁氧化还原蛋白-NADP+还原酶）的测定中具有完全活性。本文所述的表达系统适用于通过定点诱变和核磁共振光谱对菠菜铁氧化还原蛋白I进行结构-功能关系研究。
