Efficient and cost effective production of active-form human PKB using silkworm larvae.
Efficient and cost effective production of active-form human PKB using silkworm larvae.
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DOI：
10.1038/srep06016
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发表时间：
2014-08-15
影响因子：
4.6
通讯作者：
Mishima M
中科院分区：
文献类型：
--
作者：
Maesaki R;Satoh R;Taoka M;Kanaba T;Asano T;Fujita C;Fujiwara T;Ito Y;Isobe T;Hakoshima T;Maenaka K;Mishima M
蛋白激酶B（PKB）也称为Akt，参与许多信号转导途径。由于在许多人类恶性肿瘤中发现了PKB通路的改变，因此PKB被认为是癌症治疗的重要药物靶标。然而，生产足够量的活性PKB用于生物化学和结构研究是非常昂贵的，因为必须使用更高的生物表达系统来获得磷酸化的PKB。本文报道了利用家蚕核型多角体病毒（BmNPV）杆粒表达系统，以家蚕幼虫为材料，高效表达活性PKBα。重组蛋白PKB α在幼虫脂肪体中得到高效表达，并可通过GST标签进行纯化和切割。记录的最终产量约为1 mg PKBα/20只幼虫。激酶活性测定表明，重组蛋白具有较高的磷酸化活性。我们通过质谱分析进一步证实了激活环上的磷酸化。我们的结果表明，家蚕表达系统对于制备激活环磷酸化的活性形式PKBα具有价值。这种活性蛋白的有效生产将促进进一步的生物化学和结构研究，并刺激随后的药物开发。
Protein kinase B (PKB) also known as Akt is involved in many signal transduction pathways. As alterations of the PKB pathway are found in a number of human malignancies, PKB is considered an important drug target for cancer therapy. However, production of sufficient amounts of active PKB for biochemical and structural studies is very costly because of the necessity of using a higher organism expression system to obtain phosphorylated PKB. Here, we report efficient production of active PKBα using the BmNPV bacmid expression system with silkworm larvae. Following direct injection of bacmid DNA, recombinant PKBα protein was highly expressed in the fat bodies of larvae, and could be purified using a GST-tag and then cleaved. A final yield of approximately 1 mg PKBα/20 larvae was recorded. Kinase assays showed that the recombinant PKBα possessed high phosphorylation activity. We further confirmed phosphorylation on the activation loop by mass spectrometric analysis. Our results indicate that the silkworm expression system is of value for preparation of active-form PKBα with phosphorylation on the activation loop. This efficient production of the active protein will facilitate further biochemical and structural studies and stimulate subsequent drug development.
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影响因子：
2.7
作者：
Bencsik, Josef R.;Xiao, Dengming;Murray, Lesley J.
通讯作者：
Murray, Lesley J.
影响因子：
2.7
作者：
Blake, James F.;Kallan, Nicholas C.;Brandhuber, Barbara J.
通讯作者：
Brandhuber, Barbara J.
影响因子：
1.6
作者：
Fabbro, D;Batt, D;Ferrari, S
通讯作者：
Ferrari, S
影响因子：
3.7
作者：
Kato T;Thompson JR;Park EY
通讯作者：
Park EY
DOI：
10.1016/j.bbagen.2005.04.002
发表时间：
2005-10-10
影响因子：
3
作者：
Baer, K;Lisinski, I;Al-Hasani, H
通讯作者：
Al-Hasani, H