Molecular cloning, expression, and characterization of secretory phospholipase A2 in tobacco
Molecular cloning, expression, and characterization of secretory phospholipase A2 in tobacco
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DOI：
10.1007/s11745-005-1450-9
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发表时间：
2005-09-01
期刊：
影响因子：
1.9
通讯作者：
Esaka, M
中科院分区：
文献类型：
--
作者：
Fujikawa, R;Fujikawa, Y;Esaka, M
研究了烟草不同组织中磷脂酶A（2）（PLA（2））的活性。花中的PLA（2）活性比叶、茎和根中的高15倍。花中的PLA（2）活性似乎来源于Ca ~（2+）依赖性和非依赖性PLA（2）。从烟草花中分离到一个与动物分泌型PLA（2）（sPLA（2））具有同源性的蛋白质cDNA克隆，命名为Nt PLA（2）。Nt PLA（2）的cDNA编码一个127个氨基酸残基的成熟蛋白，其中含有一个30个氨基酸残基的信号肽。成熟Nt PLA（2）的氨基酸序列包含12个半胱氨酸、一个Ca 2+结合环和一个催化结构域，这些在动物sPLA（2）中通常是保守的。Nt PLA（2）mRNA主要在烟草的根和茎中表达。在大肠杆菌中表达了与硫氧还蛋白融合的重组蛋白Nt PLA（2）。从细菌细胞裂解物中，以可溶形式回收融合蛋白，并通过因子Xa蛋白酶切割。然后用离子交换层析法纯化重组Nt PLA（2）。有人发现，纯化的Nt PLA 2基本上需要Ca 2+，酶的活性时，确定使用混合胶束磷脂底物与胆酸钠的活性。以PC为底物时，Nt PLA（2）的最适pH为8-10。
Phospholipase A(2) (PLA(2)) activity was investigated in various tissues of tobacco (Nicotiana tabacum). PLA(2) activity in the flower was 15 times higher than that in the leaf, stem, and root. PLA(2) activity in the flower appears to have originated from both Ca2+-dependent and -independent PLA(2). A cDNA clone for protein with homology to animal secretory PLA(2) (sPLA(2)), denoted as Nt PLA(2), was isolated from the tobacco flower. The cDNA of Nt PLA(2) encoded a mature protein of 127 amino acid residues with a putative signal peptide of 30 residues. The amino acid sequence for mature Nt PLA(2) contains 12 cysteines, a Ca2+ binding loop, and a catalytic domain that are commonly conserved in animal sPLA(2). The Nt PLA(2) mRNA was mainly expressed in the root and stem of tobacco. The recombinant Nt PLA(2) was expressed as a fusion protein with thioredoxin in Escherichia coli. From the bacterial cell lysate, the fusion protein was recovered in soluble form and cleaved by Factor Xa proteinase. Then the recombinant mature Nt PLA(2) was purified by ion exchange chromatography. It was discovered that the purified Nt PLA2 essentially requires Ca2+, for the enzyme activity when the activity was determined using mixed-micellar phospholipid substrates with sodium cholate. The optimal activity of Nt PLA(2) was at pH 8-10 when PC was used as a substrate.