Tat核心肽段促进外源蛋白高产量及可溶性表达的机理研究

As a cell-penetrating peptide, the TAT core peptide (YGRKKRRQRRR, pI=12.8) was coded by HIV-1 virus and enriched with basic amino acid. It could deliver several heterologous macromolecules across biomembranes and without lose their bioactivity. In our pilot study for exploring its transduction function, we found interestingly that it had a novel function to promote the translation and expression of heterologous proteins in E.coli. Most suprisingly, we found that the TAT core domain was not only able to accelerate expression of heterologous proteins in E.coli, but also prompted the yield and the solubility of heterologous proteins (5-10 fold), however the mechanism was still unknown althouth we have published the first report on this issue. To elucidate it, we designed the following experiments: 1. Screening the optimal motif of TAT peptide; 2. Screening the university of TAT optimal motif for heterogeneous proteins high-yield and soluble expression; 3. Study on the mechanism of TAT optimal motif for heterogeneous proteins high-yield and soluble expression; 4. Identification of the TAT optimal motif for high-yield and soluble expression of membrane protein LSECTin and its structure analysis.This study not only provided a novel method for heterologous protein high-yield and soluble expression in prokaryotic, it also provided a new application for Tat peptide to some difficult proteins, such as membrance protein and/or disulfide bond riched proteins.

Tat核心肽段是HIV-1病毒编码的富含碱性氨基酸的穿膜肽，已知能介导多种外源物质通过各种膜性结构并保持生物活性。我们前期研究Tat核心肽段穿膜效应时，意外发现其具有促进外源蛋白高产量及可溶性表达的新功能并在国际上报道了该现象，但作用机理尚不清楚。为此，本项目拟从以下几个方面开展工作：研究Tat核心肽段促进外源蛋白高产量及可溶性表达的最优模体；研究Tat最优模体促进外源蛋白高产量及可溶性表达的普适性；研究Tat最优模体促进外源蛋白高产量及可溶性表达的作用机理；以膜蛋白LSECTin为例研究Tat核心肽段对膜蛋白高产量及可溶性表达的促进作用及其结构解析。本项目的实施将为揭示原核表达系统促进外源蛋白高产量及可溶性表达的调控模式提供新的思路及应用,而且为膜蛋白及富含二硫键等难表达蛋白体外可溶性表达富集及结构解析提供重要技术手段，具有重要的应用价值。

本项目首先使用了脑红蛋白(Ngb)评价TAT核心肽段的活性，在构建相关表达载体后，证明其具有良好的跨膜活性，比不含有TAT跨膜转导域片段的相应蛋白具有显著的保护缺氧诱导PC12细胞损伤的活性，进一步还证明了Tat-Ngb-His对PC12细胞的转导和神经保护作用。同样地，使用TAT核心肽段对跨膜蛋白（LSECtin-CRD）在大肠杆菌中的融合表达研究，也证明TAT核心肽段技术也能够促进跨膜蛋白的表达效率，为进一步拓展本项目在生物工程领域中的应用奠定了重要基础。随后对Tat核心肽段在促进原核表达体系中可溶性蛋白表达的适用范围和拓展功能的进一步研究，发现在4oC条件下即使不添加诱导剂IPTG也可促进外源蛋白的大量表达，说明4oC静置条件下Tat标签对外源蛋白表达的诱导作用是持续的累积的，为Tat功能及其在生物技术领域的应用提供了新思路。对Tat标签4oC诱导表达进行定性和定量研究，我们针对上述两种蛋白(EGFP；NGB)表达量分别进行分析，证明含Tat标签的两种蛋白表达量均远远高于未含Tat标签的宿主菌，进一步从蛋白表达量角度证明了Tat标签可诱导4oC静置条件下外源蛋白宿主菌中外源蛋白的表达。综上所述，本项目研究表明，Tat核心肽段标签可以显著促进靶蛋白的高效可溶性表达，同时本项目部分地揭示了Tat促进外源蛋白表达的机理，为后续进一步研究TAT核心肽段促进外源蛋白高产量及可溶性表达的机理、并促进其应用于实践奠定了重要基础。