Cloning, expression, and purification of a recombinant Tat-HA-NR2B9c peptide
Cloning, expression, and purification of a recombinant Tat-HA-NR2B9c peptide
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重组 Tat-HA-NR2B9c 肽的克隆、表达和纯化
DOI：
10.1016/j.pep.2012.08.011
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发表时间：
2012-10-01
影响因子：
1.6
通讯作者：
Zhu, Dong-Ya
中科院分区：
文献类型：
--
作者：
Zhou, Hai-Hui;Zhang, Ai-Xia;Zhu, Dong-Ya
为了设计一个能阻断N-甲基-D-天冬氨酸受体-2B（NR 2B）与突触后密度蛋白-95（PSD-95）相互作用的多肽，利用聚合酶链反应构建了一个编码该嵌合肽的基因片段，并将其连接到一个新的表达载体中，在一个17 RNA聚合酶表达系统中进行重组表达。该嵌合肽含有人免疫缺陷病毒1型（HIV-1）达特的细胞膜转导结构域片段、流感病毒血凝素（HA）表位标签和NR 2 B的C-末端9个氨基酸（NR 2 B 9 c）。我们将嵌合肽命名为Tat-HA-NR 2B 9 c。将含有编码Tat-HA-NR 2B 9 c的基因片段的表达质粒通过独特的酸不稳定Asp-Pro接头连接到L-天冬酰胺酶（AnsB-C）的C-末端片段。在异丙基β-D-1-硫代半乳糖苷（IPTG）诱导下，在大肠杆菌中以包涵体形式表达重组融合蛋白，经2 M尿素洗涤、4 M尿素溶解、乙醇沉淀等步骤纯化。目标嵌合肽Tat-HA-NR 2B 9 c在酸水解后从融合伴侣中释放出来，并通过等电点沉淀和超滤进行纯化。SDS-PAGE分析和MALDI-TOF-MS分析表明，纯化的Tat-HA-NR 2B 9 c具有高度的均一性。此外，我们研究了Tat-HA-NR 2B 9 c对大脑中动脉闭塞（MCAO）和再灌注大鼠缺血诱导的脑损伤的影响。并发现这种肽减少了梗死面积，改善了神经功能。(C)2012 Elsevier Inc. All rights reserved.
To design a peptide disrupting the interaction between N-methyl-D-aspartate receptors-2B (NR2B) and postsynaptic density protein-95 (PSD-95), a gene fragment encoding a chimeric peptide was constructed using polymerase chain reaction and ligated into a novel expression vector for recombinant expression in a 17 RNA polymerase-based expression system. The chimeric peptide contained a fragment of the cell membrane transduction domain of the human immunodeficiency virus type1 (HIV-1) Tat, a influenza virus hemagglutinin (HA) epitope-tag, and the C-terminal 9 amino acids of NR2B (NR2B9c). We named the chimeric peptide Tat-HA-NR2B9c. The expression plasmid contained a gene fragment encoding the Tat-HA-NR2B9c was ligated to the C-terminal fragment of L-asparaginase (AnsB-C) via a unique acid labile Asp-Pro linker. The recombinant fusion protein was expressed in inclusion body in Escherichia coli under isopropyl beta-D-1-thiogalactopyranoside (IPTG) and purified by washing with 2 M urea, solubilizing in 4 M urea, and then ethanol precipitation. The target chimeric peptide Tat-HA-NR2B9c was released from the fusion partner following acid hydrolysis and purified by isoelectric point precipitation and ultrafiltration. SDS-PAGE analysis and MALDI-TOF-MS analysis showed that the purified Tat-HA-NR2B9c was highly homogeneous. Furthermore, we investigated the effects of Tat-HA-NR2B9c on ischemia-induced cerebral injury in the rats subjected to middle cerebral artery occlusion (MCAO) and reperfusion. and found that the peptide reduced infarct size and improved neurological functions. (C) 2012 Elsevier Inc. All rights reserved.