TAT-PTD融合蛋白可能存在的跨膜递送作用机制

为探讨TAT-PTD融合蛋白的跨膜递送作用机制,采用DNA重组技术构建pGEX-TAT-GFP-表达质粒.在E.coli- BL21表达GST-TAT-GFP,并用谷胱甘肽(glutathione) Sepharose-4B亲和柱进行纯化.GST-TAT-GFP在不同条件下与细胞的作用结果表明,GST-TAT-GFP能有效进入HeLa、SMMC-7721、L-02和BEL-7402细胞,GST-TAT-GFP在递送时对时间和浓度有依赖关系.同时,温度对GST-TAT-GFP跨膜作用具有明显的影响,GST-TAT-GFP的跨膜作用受代谢抑制剂的影响很小,肝素的存在能明显抑制GST-TAT-GFP跨膜进入细胞的能力,GST-TAT-GFP对细胞活性没有影响.这些结果说明,TAT-PTD可能是通过与细胞表面的硫酸乙酰肝素等受体相结合介导融合蛋白跨膜递送进入细胞的.     

融合蛋白TAT-RIG-I-GFP的原核表达及其跨膜递送

本研究旨在探讨融合蛋白TAT-RIG-I-GFP原核表达载体的构建并验证TAT在跨膜递送中的作用。首先设计了4对特异性引物,克隆了绿头鸭AnasplatyrhynchosRIG-I基因,构建了pET-TAT-RIG-I-GFP和pET-RIG-I-GFP原核表达载体;转化至感受态DE3细胞,经IPTG诱导表达,利用His60镍亲和层析柱纯化,进行SDS-PAGE;然后,将纯化后的上述两种表达蛋白分别孵育DF-1细胞;最后利用荧光显微镜观察是否在DF-1细胞产生相应的荧光。结果证实,携带有TAT的pET-TAT-RIG-I-GFP融合蛋白在DF-1细胞中显示出明显的绿色荧光;而不具有TAT的pET-RIG-I-GFP蛋白却不能显示绿色荧光。这表明携带TAT的融合蛋白已成功进入DF-1细胞,并在跨膜递送过程中发挥了关键作用。上述为进一步研制家禽的抗病毒药物奠定了基础。     

HIV-1 TAT蛋白转导肽的研究进展

TAT蛋白转导肽是人类免疫缺陷病毒1型(human immunodeficiency virus type 1, HIV-1)编码的一段富含碱性氨基酸、带正电荷的多肽,属于蛋白转导域家族的一员。长期研究发现其全长及11个碱性氨基酸富集区的核心肽段(YGRKKRRQRRR)不仅能够在包括蛋白质、多肽及核酸等多种外源生物大分子的跨膜转导过程中具有重要作用,而且能够携带这些外源生物大分子通过活体细胞的各种生物膜性结构(如细胞膜和血脑屏障等)并发挥生理功能,但其跨膜转导机制仍不明确。新近研究还发现TAT核心肽段在促进外源蛋白高效表达过程中也具有重要作用,能够显著增加外源蛋白高效、可溶性表达的水平,显示了TAT蛋白转导肽的新功能。以TAT蛋白转导肽跨膜转导作用的长期研究背景为基础,分别从TAT蛋白转导肽的结构特点、其跨膜转导作用的影响因素及其作用机制等方面进行了系统综述,进一步结合TAT蛋白转导肽的最新研究进展分别从药物研发、机制探索及新功能的开发等方面展望了后续研究方向与应用价值,不仅为深入阐述TAT蛋白转导肽的跨膜转导作用的功能意义提供了参考依据,而且为TAT蛋白转导肽在微生物工程及蛋白质工程等领域的潜在应用价值提供了重要参考信息。     

TAT蛋白转导肽介导的秀丽线虫体内外源蛋白的跨膜转导研究

TAT蛋白转导肽是HIV-1病毒编码的一段富含碱性氨基酸序列的多肽,能够高效介导多种外源生物大分子通过多种膜性结构,如细胞质膜和血脑屏障等。为探索TAT蛋白转导肽介导的秀丽线虫体内外源蛋白跨膜转导作用,以EGFP为报告基因结合常规分子克隆技术构建了原核表达载体pET28b-EGFP和pET28-TAT-EGFP,继而利用诱导剂IPTG(终浓度1mmol/L)诱导表达了靶蛋白并结合荧光显微观察、SDS-PAGE和Western blot等鉴定技术获得表达靶蛋白的大肠杆菌BL21(DE3)细胞,最后将其涂布到含有Kana⁺的LB固体培养基上直接饲喂野生型N2株系线虫,利用荧光显微镜观察绿色荧光信号在线虫体内的分布。结果证明,TAT-EGFP融合蛋白较之于EGFP可高效、可溶性表达,而且通过直接饲喂秀丽线虫表达靶蛋白的大肠杆菌48小时后,TAT-EGFP荧光信号明显分布于线虫肠壁细胞,而EGFP荧光信号则分布在秀丽线虫肠腔,空载体对照组未见任何荧光信号,说明TAT蛋白转导肽能够高效介导外源蛋白在秀丽线虫体内跨膜转导。同时,通过比较空载体对照组与实验组线虫微分干涉图像,未见线虫出现明显的细胞形态变化,说明TAT蛋白转导肽介导的外源蛋白跨膜转导作用是安全的,为在秀丽线虫体内直接研究外源蛋白的功能以及进行蛋白药物的研发提供了重要参考。     

蛋白转导域透膜作用的研究进展

HIV-TAT蛋白转导域（Protein transduction domain,,PTD）是新近发现的一种在蛋白转导过程中能高效穿过生物膜的结构域,源自人类免疫缺陷病毒Tat蛋白的一段碱性氨基酸多肽,能与多肽、蛋白质及DNA等分子连接并跨膜导入绝大部分的组织细胞或透过血脑屏障,转导效率高且对细胞无损伤。TAT-PTD与细胞膜之间的电荷作用,吸附于膜表面,依赖脂筏介导的巨胞饮作用进入细胞。TAT融合蛋白系统是一种极有价值的运载工具,在基础医学研究和临床治疗方面有着广泛的应用前景。     

带有蛋白转导结构域的Bcr/Abl癌蛋白片段的表达及其跨膜转运

HIV 1编码的反式激活蛋白TAT具有将细胞外蛋白转导进入细胞的基序 ,称为蛋白转导结构域 (PTD) .为研究PTD介导的PTD Bcr Abl融合蛋白的跨膜转运 ,合成了编码PTD的基因片段 ,并与PCR扩增的慢性粒细胞白血病癌蛋白bcr abl基因片段融合 .在大肠杆菌中表达纯化了融合蛋白 ,将纯化的融合蛋白加入培养的HL60细胞和C2C12细胞后 ,发现PTD基序可以介导Bcr Abl蛋白自由从细胞外跨膜转导进入细胞内 .研究结果可能为用外源蛋白负载 (loading)免疫活性细胞如抗原提呈细胞提供新的途径 .     

多肽TAT与核定位信号介导的蛋白质入核递送

增强型绿色荧光蛋白与蛋白质转导结构域TAT、SV40大T抗原的核定位信号以融合蛋白的形式在大肠杆菌中表达 ,纯化后转导A431细胞 ,大部分细胞核内都可以观察到绿色荧光 ,说明TAT NLS可以有效介导蛋白质的入核递送。这种蛋白质递送系统可望用于转录治疗等研究领域。     

穿膜肽TAT和R9的生物学效应的体内体外研究

本文比较了两种常用穿膜肽TAT和R9的体外穿膜效率、对细胞影响以及小鼠活体内不同器官的穿膜效率。非特异性穿膜肽TAT和R9分别为11个氨基酸和9个氨基酸的短肽,能够有效地穿过细胞膜进入细胞。本研究纯化制备出融合蛋白TAT-EGFP及R9-EGFP多肽,用相同浓度的活性TAT-EGFP和R9-EGFP处理肺癌细胞,观察比较二者的穿膜活性及效率;设置不同浓度梯度检测两种穿膜肽是否对细胞产生影响;通过小鼠腹腔注射TAT-EGFP和R9-EGFP活性肽段,研究二者在活体能够穿膜到达的靶位置,并比较分析二者穿膜的效果器官分布。结果显示制备获得的两种融合蛋白TAT-EGFP和R9-EGFP均能有效穿过实现穿膜功能,两种穿膜肽对乳腺癌细胞影响较小。活体试验证明两种穿膜肽在心、肝、脾、肺、肾器官均有分布,R9主要富集在小鼠的肾和肝脏中,TAT在6种被研究的器官都有富集,两种细胞穿膜肽都能透过血脑屏障到达脑部。相对来说,TAT在小鼠活体中的穿膜效果更好,荧光富集多,R9整体穿膜效果较弱。本研究分析比较了两种穿膜肽的细胞安全性浓度和活体的穿膜能力,为TAT和R9两种穿膜肽在后续试验中的合理有效的选择和使用,提供了基础数据和指导性依据。     

TAT蛋白介导外源物质进入细胞的作用机制探讨

来源于人类免疫缺陷病毒HIV-1的反式激活因子(trans-activator,TAT)蛋白能够有效的介导多肽、蛋白质、基因以及一些其它的物质进入细胞。它以低亲和力与细胞上的受体相结合,通过破坏细胞质膜,以非内吞途径转导外源物质进入细胞内部。     

TAT-EDAG融合蛋白的原核表达及其转导活性的研究

HIV-TAT蛋白转导域（PTD）是新近发现的一种在蛋白转导过程中能高效穿过生物膜的结构域,它能将与之连接的多肽、蛋白质及DNA等分子跨膜导入几乎所有的组织和细胞,转导效率高而对细胞没有损伤.构建了TAT-EDAG、TAT-GFP融合蛋白原核表达载体,在大肠杆菌BL21（DE3）细胞中实现了两种融合蛋白的可溶性原核表达,在非变性条件下进行蛋白纯化,获得了纯度在90%以上的融合蛋白.脱盐处理后,利用TAT-GFP转染体外培养的鼠成纤维细胞证实了TAT转导肽的生物活性;利用TAT-EDAG转染体外培养的HL-60细胞,Western blotting分析表明：TAT-EDAG可以导入HL-60细胞中.这为下一步应用于体外造血干细胞扩增研究奠定了基础.     

Identification and characterization of a novel cell-penetrating peptide of 30Kc19 protein derived from Bombyx mori

Cell-penetrating peptides (CPPs) or protein transduction domains (PTDs) have attracted increasing attention due to their high potential to deliver various, otherwise impermeable, bioactive agents, such as drugs and proteins across cell membranes. A number of CPPs have been discovered since then. Recently, 30Kc19 protein has attracted attention because it was the first cell-penetrating protein that has been found in insect hemolymph. Here, we report a cell-penetrating peptide derived from 30Kc19 protein, VVNKLIRNNKMNC, which efficiently penetrates cells when supplemented to medium for mammalian cell culture. Moreover, like other CPPs, this “Pep-c19” also efficiently delivered cell-impermeable cargo proteins, such as green fluorescent protein (GFP) into cells. In addition to the in vitro system, Pep-c19 exhibited the cell-penetrating property in vivo. When Pep-c19 was intraperitoneally injected into mice, Pep-c19 successfully delivered cargo proteins into various organ tissues with higher efficiency than the 30Kc19 protein itself, and without toxicity. Our data demonstrates that Pep-c19 has a great potential as a cell-penetrating peptide that can be used as a therapeutic tool to efficiently deliver different cell-impermeable cargo molecules into the tissues of various organs.     

TAT蛋白转导域：蛋白质治疗的新曙光

TAT蛋白转导域是源自人类免疫缺陷病毒Tat蛋白的一段碱性氨基酸多肽,能够将与之共价连接的多肽、蛋白、核酸等生物大分子快速而高效地转导入细胞内部,在药物转运和疾病治疗等领域有着巨大的应用潜力.TAT蛋白转导域首先通过电荷相互作用吸附于细胞膜,然后通过脂筏介导的巨胞饮作用进入细胞.随着体外研究的不断成熟,应用TAT蛋白转导域治疗人类肿瘤、卒中、炎症等疾病的动物模型也获得了成功,TAT蛋白转导域进入临床指日可待.     

Cry Protein Crystals: A Novel Platform for Protein Delivery

Protein delivery platforms are important tools in the development of novel protein therapeutics and biotechnologies. We have developed a new class of protein delivery agent based on sub-micrometer-sized Cry3Aa protein crystals that naturally form within the bacterium Bacillus thuringiensis. We demonstrate that fusion of the cry3Aa gene to that of various reporter proteins allows for the facile production of Cry3Aa fusion protein crystals for use in subsequent applications. These Cry3Aa fusion protein crystals are efficiently taken up and retained by macrophages and other cell lines in vitro, and can be delivered to mice in vivo via multiple modes of administration. Oral delivery of Cry3Aa fusion protein crystals to C57BL/6 mice leads to their uptake by MHC class II cells, including macrophages in the Peyer’s patches, supporting the notion that the Cry3Aa framework can be used to stabilize cargo protein against degradation for delivery to gastrointestinal lymphoid tissues.     

Induction of antigen-specific CTL by recombinant HIV trans-activating fusion protein-pulsed human monocyte-derived dendritic cells

Several systems have been tested for introduction of Ags into human dendritic cells (DC). Most of them to date, however, are complex and possess limited efficiency. Recent advances in HIV trans-activating (TAT) fusion protein technology permit extremely high transduction efficiencies for a majority of mammalian cell types. Here we report our attempts to develop a simple, but highly efficient, protocol for loading of antigenic protein into DC using TAT fusion technology. A TAT-minigene fusion protein was generated, encoding both the HLA-A2-restricted influenza matrix protein-derived epitope (GILVFTFTL, Flu-M1) and a melanoma Ag gp100-derived modified epitope (YLEPGPVTV, G9(280)-9V). In addition, both a TAT-Her2/neu extracellular domain (ECD) fusion protein and a TAT-green fluorescence protein fusion protein were generated. Over 95% of DC stained positively for TAT-green fluorescence protein within 20 min of coculture. DC treated with TAT-minigene were efficiently recognized by both Flu-M1 and G9(280)-9V-specific T cells in cytotoxicity assays and IFN-gamma ELISPOT assays. In contrast, DC pulsed with minigene fusion protein lacking TAT were either poorly recognized or not recognized by the T cells. DC pulsed with TAT-minigene also efficiently induced Flu-M1-specific T cells from naive lymphocytes. Similarly, DC treated with TAT-Her2/neu ECD stimulated patient-derived lymphocytes that specifically recognized Her2/neu(+) ovarian and breast cancer cell lines. The CTL induced by TAT-Her2/neu ECD-pulsed DC specifically recognized the Her2/neu ECD-derived immunogenic peptide E75 (KIFGSLAFL). Our data suggest that TAT fusion proteins efficiently transduce DC and induce Ag-specific T cells. This could prove to be a useful method for treatment of infectious diseases and cancer.     

Tat 蛋白的PTD区段促进GFP蛋白进入骨髓瘤细胞SP2/0

随着生物工程技术的迅速发展 ,多肽与蛋白质类药物的增长速度相当可观 ,可是这些药物常因受到各种因素的影响而疗效偏低 ,其中生物膜的屏障作用是主要因素之一。近年来发现一种来源于人类免疫缺陷病毒ＨＩＶ 1Ｔａｔ(Ｔｒａｎｓ ａｃｔｉｖａ ｔｏｒ)蛋白的蛋白功能区 ,称之为ＰＴＤ区段 (Ｐｒｏｔｅｉｎｔｒａｎｓｄｕｃｔｉｏｎｄｏｍａｉｎ ,ＹＧＲＫＫＲＲＱＲＲＲ)的〔1 ,2〕,能够有效引导肽段或者蛋白质进入细胞 ,具有蛋白传送的功能〔3〕。 1988年Ｍａｕｒｉｃｅ和Ｐａｕｌ发现Ｔａｔ蛋白能够穿过细胞膜〔4〕 ;1994年Ｓｔｅｐｈｅｎ…     

TAT-Cloan-DH-EGFP融合蛋白的原核表达及其在舞毒蛾幼虫体内的跨膜转导

【目的】为了探究滞育激素经昆虫取食的生物效应, 需设计一种能够加速跨膜运输的融合蛋白, 克服经昆虫消化道的降解作用。【方法】本研究以分月扇舟蛾Clostera anastomosis滞育激素基因Cloan-DH和TAT穿膜肽编码序列为基础, 构建TAT-Cloan-DH融合基因; 然后以增强型绿色荧光蛋白（enhanced green fluorescent protein, EGFP）为标签, 构建在pET-22b载体中。在0.2 mmol/L IPTG诱导下, 在大肠杆菌Escherichia coli BL21 Rosetta(DE3)中表达出TAT-Cloan-DH-EGFP融合蛋白。 再分别将表达有TAT-Cloan-DH-EGFP和EGFP融合蛋白的大肠杆菌BL21菌体拌入人工饲料,经舞毒蛾Lymantria dispar幼虫持续取食0, 8, 24, 48, 72和90 h后, 随机挑选试虫进行组织固定并制作石蜡切片, 选取中肠所在体段切片进行组织荧光分析。【结果】37℃ 0.2 mmol/L IPTG诱导条件下, 表达的TAT-Cloan-DH-EGFP融合蛋白的分子量为61 kD, 主要以包涵体形式表达, 融合蛋白约占细胞总蛋白含量的18.1%; 随着试虫取食含TAT-Cloan-DH-EGFP融合蛋白饲料的增加, 虫体组织的绿色荧光强度逐渐增加, 取食72 h后, 组织荧光强度达到最高。【结论】实验结果说明融合蛋白TAT-Cloan-DH-EGFP可以通过消化道横向跨膜运输, 到达虫体其他组织, 且该过程与蛋白的摄入量成比例。     

Cytoplasmic transduction peptide (CTP): new approach for the delivery of biomolecules into cytoplasm in vitro and in vivo

The protein transduction domain (PTD) of HIV-1 TAT has been extensively documented with regard to its membrane transduction potential, as well as its efficient delivery of biomolecules in vivo. However, the majority of PTD and PTD-conjugated molecules translocate to the nucleus rather than to the cytoplasm after transduction, due to the functional nuclear localization sequence (NLS). Here, we report a cytoplasmic transduction peptide (CTP), which was deliberately designed to ensure the efficient cytoplasmic delivery of the CTP-fused biomolecules. In comparison with PTD, CTP and its fusion partners exhibited a clear preference for cytoplasmic localization, and also markedly enhanced membrane transduction potential. Unlike the mechanism underlying PTD-mediated transduction, CTP-mediated transduction occurs independently of the lipid raft-dependent macropinocytosis pathway. The CTP-conjugated Smac/DIABLO peptide (Smac-CTP) was also shown to be much more efficient than Smac-PTD in the blockage of the antiapoptotic properties of XIAP, suggesting that cytoplasmic functional molecules can be more efficiently targeted by CTP-mediated delivery. In in vivo trafficking studies, CTP-fused beta-gal exhibited unique organ tropisms to the liver and lymph nodes when systemically injected into mice, whereas PTD-beta-gal exhibited no such tropisms. Taken together, our findings implicate CTP as a novel delivery peptide appropriate for (i) molecular targeting to cytoplasmic compartments in vitro, (ii) the development of class I-associated CTL vaccines, and (iii) special drug delivery in vivo, without causing any untoward effects on nuclear genetic material.