RECONSTITUTION OF THE SPINACH OXYGEN-EVOLVING COMPLEX WITH RECOMBINANT ARABIDOPSIS MANGANESE-STABILIZING PROTEIN
RECONSTITUTION OF THE SPINACH OXYGEN-EVOLVING COMPLEX WITH RECOMBINANT ARABIDOPSIS MANGANESE-STABILIZING PROTEIN
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DOI：
10.1007/bf00039525
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发表时间：
1994-10-01
影响因子：
5.1
通讯作者：
YOCUM, CF
中科院分区：
文献类型：
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作者：
BETTS, SD;HACHIGIAN, TM;YOCUM, CF
蓝藻、绿藻和高等植物的psbO基因编码33 kDa的锰稳定蛋白（MSP）前体，MSP是光系统II （PSII）的水溶性亚基。利用pET-T7克隆/表达系统，在大肠杆菌中表达了拟南芥psbO的全长cDNA克隆。经诱导后，在强曝气条件下生长的细胞中积累了高水平的前体蛋白。在弱曝气条件下生长的细胞中，成熟蛋白通过诱导积累。在中等曝气条件下生长的细胞中，随着诱导光密度的增加，前体细胞与成熟MSP的比例降低。这两种形式的蛋白质作为包涵体积累，成熟的蛋白质可以在不释放前体的温和变性条件下从包涵体中释放出来。与分离菠菜MSP相比，再生拟南芥MSP在恢复菠菜中MSP缺失的PSII膜的O-2进化活性方面的效果为87%；然而，外源蛋白与菠菜PSIIs结合的亲和力约为原生蛋白的一半。我们还报道了先前发表的拟南芥psbO DNA序列的更正（Ko et al., Plant Mol Biol 14(1990) 217-227）。
The psbO gene of cyanobacteria, green algae and higher plants encodes the precursor of the 33 kDa manganese-stabilizing protein (MSP), a water-soluble subunit of photosystem II (PSII). Using a pET-T7 cloning/expression system, we have expressed in Escherichia coli a full-length cDNA clone of psbO from A rabidopsis thaliana. Upon induction, high levels of the precursor protein accumulated in cells grown with vigorous aeration. In cells grown under weak aeration, the mature protein accumulated upon induction. In cells grown with moderate aeration, the ratio of precursor to mature MSP decreased as the optical density at induction increased. Both forms of the protein accumulated as inclusion bodies from which the mature protein could be released under mildly denaturing conditions that did not release the precursor. Renatured Arabidopsis MSP was 87% as effective as isolated spinach MSP in restoring O-2 evolution activity to MSP-depleted PSII membranes from spinach; however, the heterologous protein binds to spinach PSIIs with about half the affinity of the native protein. We also report a correction to the previously published DNA sequence of Arabidopsis psbO (Ko et al., Plant Mol Biol 14 (1990) 217-227).