蛋白激酶D1催化结构域在昆虫细胞/杆状病毒表达系统内的表达、纯化和活性测定

Expression, purification and characterization of catalytic domain of protein kinase D1 in baculovirus-insect cell expression system

摘要:

蛋白激酶D (Protein kinase D, PKD) 是一种新的丝氨酸/苏氨酸蛋白激酶家族和甘油二酯 (Diacylglycerol, DAG) 受体，参与细胞内多种生理生化过程。为获得高纯度的PKD1的催化结构域 (PKD1-cat) 用于晶体学结构的研究，将带有GST标签的PKD1-cat基因克隆到杆状病毒转移载体pFastBac1中，构建了重组质粒。将重组质粒转化到含穿梭载体Bacmid的DH10Bac感受态细胞中，转座后获得了含目的基因GST-PKD1-cat的重组Bacmid。重组Bacmid DNA转染Sf9昆虫细胞后，获得重组杆状病毒并扩毒。将毒种以5 PFU/cell的感染复数感染悬浮培养的T.ni昆虫细胞，SDS-PAGE和Western blotting检测表达产物。结果显示，表达产物在分子量约68 kDa处有一特异条带可与GST单克隆抗体发生反应。经谷胱甘肽琼脂糖凝胶亲和层析纯化和 PreScission Protease 切除GST标签后，得到了纯度很高的分子量约42 kDa的目的蛋白PKD1-cat。体外PKD激酶活性实验结果显示，随着PKD1-cat浓度的增加，激酶活性增高。这些结果显示截短的重组PKD1-cat有很高的催化活性和纯度，为采用核磁共振或晶体学方法解析PKD1-cat的三维结构奠定了基础。

Abstract:

Protein kinase D (PKD) is a novel family of serine/threonine kinases and diacylglycerol (DAG) receptors and has been documented in a variety of cellular processes. To get high purity catalytic domain of PKD1 (PKD1-cat) for crystallography study, the GST-tagged PKD1-cat gene was cloned into a baculovirus transfer vector pFastBac1 (donor plasmid). When the recombinant plasmid was transformed into DH10Bac competent Escherichia coli, which contains a baculovirus shuttle vector (bacmid), transposition occurs to generate a recombinant bacmid containing the gene of interest (GST-PKD1-cat). The recombinant bacmid DNA was transfected into Spodoptera frugiperda Sf9 insect cells to generate recombinant baculovirus, which was then amplified through multiple rounds of infection in Sf9 cells. After that, Trichoplusia ni insect cells in suspension culture were infected with baculoviral stock at a multiplicity of infection (MOI) of 5 PFU/cell. SDS-PAGE and Western blotting analysis confirmed the detection of a 68 kDa protein by the glutathione S-transferase (GST) monoclonal antibody. The recombination protein was purified by Glutathione sepharose affinity chromatography and cleaved by PreScission Protease to remove GST tag, and a highly pure 42 kDa protein which was consistent with the molecular weight of the expected PKD1-cat protein was detected on SDS-PAGE. The activity of purified PKD1-cat protein was determined by in vitro PKD kinase assay. Our data showed that the kinase activity increased with the concentration of purified PKD1-cat protein. These results showed that the truncated recombinant PKD1-cat protein was highly active and pure, and could potentially be used for solving 3D structure of this protein by Nuclear Magnetic Resonance (NMR) or crystallography.