Efficient production of recombinant PP2A at a low temperature using a baculovirus expression system.
Efficient production of recombinant PP2A at a low temperature using a baculovirus expression system.
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DOI：
10.1016/j.btre.2016.07.004
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发表时间：
2016-09
期刊：
影响因子：
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通讯作者：
Yasumoto, Takeshi
中科院分区：
文献类型：
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作者：
Ikehara, Tsuyoshi;Nakashima, Shihoko;Nakashima, Junichi;Kinoshita, Tsubasa;Yasumoto, Takeshi
利用杆状病毒表达系统，在High Five昆虫细胞中表达重组人PP2A催化亚基（rhPP2Ac）。在19 °C下的表达可以比在27 °C下进行的孵育产生具有更高活性和更大量的rhPP2Ac。为了检测低温表达对其他磷酸酶的影响，我们在High Five昆虫细胞中表达了人PP2B和PP2C。优化杆状病毒系统中的表达温度对于生产重组蛋白是有效的。蛋白磷酸酶2A（PP2A）是用于检测以冈田酸和微囊藻毒素为代表的几种天然毒素的酶。我们发现，在High Five昆虫细胞中，当细胞在19 °C而不是在常规条件下使用的27 °C下培养时，重组人PP2A催化亚基（rhPP2Ac）的产量可以显著增加。酶的产率和纯度分别提高了4倍和3倍。使用重组人蛋白磷酸酶2B观察到改变培养温度的益处，但未观察到2Cα。不同的酶之间的反应表明参与的酶特异性机制，导致催化亚基过表达。这是第一份在低于High Five昆虫细胞杆状病毒表达系统中使用的常规培养条件（27 °C）的温度下生产rhPP2Ac的报告。
The recombinant human PP2A catalytic subunit (rhPP2Ac) was produced in the baculovirus expression system with High Five insect cells. The expression at 19 °C can produce the rhPP2Ac with a higher activity and in a larger quantity than in the incubation conducted at 27 °C. To examine the effects of the low temperature expression on other phosphatases, we expressed human PP2B and PP2C in High Five insect cells. Optimizing the expression temperature in a baculovirus system is effective for producing a recombinant protein. Protein phosphatase 2A (PP2A) is an enzyme useful for detecting several natural toxins represented by okadaic acid and microcystins. We found that the production of the recombinant human PP2A catalytic subunit (rhPP2Ac) in High Five insect cells could markedly increase when the cells were cultured at 19 °C instead of 27 °C used under conventional conditions. The yield and purity of the enzyme increased four- and three-folds, respectively. The benefit of the altered culturing temperature was observed with the recombinant human protein phosphatase 2B but not 2Cα. The different responses among the enzymes suggest the involvement of an enzyme-specific mechanism that leads to the catalytic subunit overexpression. This is the first report to produce rhPP2Ac at a temperature lower than that used under conventional culture conditions (27 °C) used in the baculovirus expression system with High Five insect cells.