An assay for phosphoinositide phosphatases utilizing fluorescent substrates
An assay for phosphoinositide phosphatases utilizing fluorescent substrates
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DOI：
10.1006/abio.2001.5179
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发表时间：
2001-08-01
影响因子：
2.9
通讯作者：
Dixon, JE
中科院分区：
文献类型：
--
作者：
Taylor, GS;Dixon, JE
在大肠杆菌BL 21（DE 3）Codon Plus细胞（Stratagene，拉霍亚，CA）中用C末端六组氨酸标签表达MTM 1。使用含有5-NheI和3-SalI限制性连接子的寡核苷酸引物，通过PCR扩增含有MTM 1开放阅读框而无终止密码子的cDNA片段。将该片段插入用NheI和XhoI消化的pET 21 a载体中，以产生pET-MTM 1-H6。如Sac 1 p融合蛋白所述表达和纯化His标记的MTM 1（1）。如前所述表达和纯化细菌重组PTEN（2）。
(MTM1) was expressed with a C-terminal six-histidine tag in Escherichia coli BL21 (DE3) Codon Plus cells (Stratagene, La Jolla, CA). A cDNA fragment containing the MTM1 open-reading frame without a stop codon was amplified by PCR using oligonucleotide primers containing 5-NheI and 3-SalI restriction linkers. This fragment was inserted into pET21a vector digested with NheI and XhoI to create pET-MTM1-H6. His-tagged MTM1 was expressed and purified as described for the Sac1p fusion protein (1). Bacterial recombinant PTEN was expressed and purified as previously described (2).