Recombinant expression, purification and characterization of Bombyx mori (Lepidoptera: Bombycidae) pyridoxal kinase
Recombinant expression, purification and characterization of Bombyx mori (Lepidoptera: Bombycidae) pyridoxal kinase
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家蚕（鳞翅目：家蚕科）吡哆醛激酶的重组表达、纯化和表征
DOI：
10.14411/eje.2011.003
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发表时间：
2011-01
影响因子：
1.3
通讯作者：
Zhang, Ping-Ping
中科院分区：
文献类型：
--
作者：
Xie, Yan-Feng;Zhang, Jian-Yun;Ma, Wang;Huang, Shuo-Hao;Huang, Long-Quan;Zhang, Ping-Ping
吡哆醛激酶（Pyridoxal kinase， PLK; EC 2.7.1.35）是家蚕代谢维生素B6 （VB6）的关键酶。采用亚克隆技术构建融合表达载体pET-22b-BPLK-His，在大肠杆菌中表达重组家蚕PLK，并进行纯化和鉴定。利用生物信息学方法推断该酶的蛋白质结构和基因组组织。利用Ni Sepharose亲和柱层析，重组蛋白的纯度非常高（约90%）。重组PLK具有较高的特异性酶活性（1800 nmol/min/mg蛋白）。该酶在较窄的pH范围内（5.5-6.0）具有最大的催化活性，在饱和底物浓度下，zn2 +是最有效的催化阳离子。当只有三乙醇胺作为阳离子存在时，K +是PLK的活化剂。初速度的双倒易图表明该酶是通过顺序催化机制催化反应的。在最佳条件下，ATP和吡哆醛底物的Km值分别为57.9±5.1和44.1±3.9µM。家蚕基因组包含一个PLK基因拷贝，全长7.73 kb，包含5个外显子和4个内含子，位于第8条染色体上。在天然条件下，PLK可能是具有两个相同亚基的二聚体，并且假设每个单体包含8个D -螺旋（d1 -8）， 9个e -链（E1-9）和两个310螺旋的片段。
Pyridoxal kinase (PLK; EC 2.7.1.35) is a key enzyme in the metabolism of vitamin B6 (VB6) in Bombyx mori. A fusion expressional vector pET-22b-BPLK-His was constructed using a sub-cloning technique, the recombinant B. mori PLK was then expressed in Escherichia coli, purified and characterized. Bioinformatics were used to deduce the protein structure and genomic organization of this enzyme. Using Ni Sepharose affinity column chromatography, the recombinant protein was purified to very high degree (approximately 90%). The recombinant PLK exhibits a high specific enzymatic activity (1800 nmol/min/mg of protein). The maximum catalytic activity of this enzyme was recorded over a narrow pH range (5.5-6.0) and Zn 2+ is the most effective cation for catalysis under saturating substrate concentrations. When only triethanolamine is present as the cation, K + is an activator of PLK. A double reciprocal plot of initial velocity suggests that the enzyme catalyses the reaction by means of a sequential catalytic mecha- nism. Under optimal conditions, the Km value for the substrates of ATP and pyridoxal are 57.9 ± 5.1 and 44.1 ± 3.9 µM. B. mori's genome contains a single copy of the PLK gene, which is 7.73 kb long and contains five exons and four introns, and is located on the eighth chromosome. The PLK may be a dimer with two identical subunits under native conditions, and it is hypothesized that each monomer contains eight D -helices (D 1-8), nine E-strands (E1-9) and two segments of 310 helices.
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影响因子：
5.6
作者：
M. Torruella;M. Flawiá;C. Eisenschlos;L. M. Vedia;C. Rubinstein;H. Torres
通讯作者：
M. Torruella;M. Flawiá;C. Eisenschlos;L. M. Vedia;C. Rubinstein;H. Torres
DOI：
10.1093/jn/114.9.1664
发表时间：
1984-09
期刊：
The Journal of nutrition
影响因子：
--
作者：
Alfred H. Merrill;J. Henderson;Elaine Wang;Bill W. McDonald;W. Millikan
通讯作者：
Alfred H. Merrill;J. Henderson;Elaine Wang;Bill W. McDonald;W. Millikan
DOI：
10.1023/a:1021079110358
发表时间：
1999-04
期刊：
Journal of Protein Chemistry
影响因子：
--
作者：
B. Maras;S. Valiante;S. Orrù;M. Simmaco;D. Barra;J. Churchich
通讯作者：
B. Maras;S. Valiante;S. Orrù;M. Simmaco;D. Barra;J. Churchich
影响因子：
2.1
作者：
Y. Yang;G. Zhao;M. Winkler
通讯作者：
Y. Yang;G. Zhao;M. Winkler
影响因子：
4.3
作者：
H. Lum;F. Kwok;S. Lo
通讯作者：
H. Lum;F. Kwok;S. Lo