Anderca Marinela Ioana, Furuichi Takuya, Pinontoan Reinhard, Muto Shoshi
Graduate School of Bioagricultural Sciences, Nagoya University, Chikusa-ku, Nagoya, 464-8601 Japan.
Plant Cell Physiol. 2002 Nov;43(11):1276-84. doi: 10.1093/pcp/pcf155.
We isolated a full-length cDNA encoding a nucleoside diphosphate (NDP) kinase from a Dunaliella tertiolecta cDNA library by homology cloning and rapid amplification of cDNA ends-PCR. The cDNA sequence, consisting of 840 bp, contained an open reading frame coding for a 221-amino acid protein. The predicted 24-kDa protein was named DtNDK1. It possesses all the residues involved in nucleotide binding and catalysis and, in its long N-terminus, contains putative mitochondrial targeting peptides. The full-length pre-protein expressed in Escherichia coli as a recombinant N-terminally His-tagged protein was retained in inclusion bodies, totally devoid of NDP kinase activity. Upon expression in yeast cells, the full-length protein His-tagged at the C-terminus was found processed in a soluble form that was lacking the first 67 amino acids from the N-terminus. The mature protein, which was purified by affinity chromatography to near homogeneity, showed NDP kinase activity. Confocal microscopy on yeast cells expressing the recombinant protein revealed the specific mitochondrial localization of DtNDK1 labeled at the C-terminus with green fluorescent protein.
我们通过同源克隆和cDNA末端快速扩增PCR技术，从杜氏盐藻（Dunaliella tertiolecta）cDNA文库中分离出一个编码核苷二磷酸（NDP）激酶的全长cDNA。该cDNA序列长840 bp，包含一个编码221个氨基酸蛋白质的开放阅读框。预测的24 kDa蛋白被命名为DtNDK1。它具有所有参与核苷酸结合和催化的残基，并且在其长的N末端包含假定的线粒体靶向肽。在大肠杆菌中作为重组N末端His标签蛋白表达的全长前体蛋白保留在包涵体中，完全没有NDP激酶活性。在酵母细胞中表达时，发现C末端带有His标签的全长蛋白以可溶形式加工，该形式缺少N末端的前67个氨基酸。通过亲和层析纯化至近乎均一的成熟蛋白显示出NDP激酶活性。对表达重组蛋白的酵母细胞进行共聚焦显微镜检查，发现用绿色荧光蛋白标记C末端的DtNDK1具有特定的线粒体定位。
