Króliczewski Jaroslaw, Hombek-Urban Katarzyna, Szczepaniak Andrzej
The Institute of Biochemistry and Molecular Biology, University of Wroclaw, Przybyszewskiego 63/77, 51-148 Wroclaw, Poland.
Biochemistry. 2005 May 24;44(20):7570-6. doi: 10.1021/bi047422w.
An overexpression system for spinach apocytochrome b(6) as a fusion protein to a maltose-binding protein in Escherichia coli was established using the expression vector pMalp2. The fusion of the cytochrome b(6) to the periplasmic maltose-binding protein directs the cytochrome on the Sec-dependent pathway. The cytochrome b(6) has a native structure in the bacterial cytoplasmic membrane with both NH(2) and COOH termini on the same, periplasmic side of the membrane but has the opposite orientation compared to that in thylakoid. Our data also show that in the E. coli cytoplasmic membrane, apocytochrome b(6) and exogenic hemes added into a culture media spontaneously form a complex with similar spectroscopic properties to native cytochrome b(6). Reconstituted membrane-bound cytochrome b(6) contain two b hemes (alpha band, 563 nm; average E(m,7) = -61 +/- 0.84 and -171 +/- 1.27 mV).
利用表达载体pMalp2在大肠杆菌中建立了一种菠菜脱辅基细胞色素b(6)作为与麦芽糖结合蛋白的融合蛋白的过表达系统。细胞色素b(6)与周质麦芽糖结合蛋白的融合将细胞色素导向依赖Sec的途径。细胞色素b(6)在细菌细胞质膜中具有天然结构，其NH(2)和COOH末端位于膜的同一周质侧，但与类囊体中的方向相反。我们的数据还表明，在大肠杆菌细胞质膜中，脱辅基细胞色素b(6)和添加到培养基中的外源性血红素自发形成一种具有与天然细胞色素b(6)相似光谱特性的复合物。重组的膜结合细胞色素b(6)含有两个b型血红素（α带，563 nm；平均E(m,7)=-61±0.84和-171±1.27 mV）。
