TAT蛋白转导结构域介导融合蛋白在小鼠活体的跨膜递送作用

目的探讨跨膜递送短肽——TAT蛋白转导结构域(简称TAT)介导的与其融合的活性蛋白在活体的跨膜递送作用。方法以融合蛋白GST-TAT-GFP,GST-GFP-TAT和GST-GFP为研究模型蛋白,不经过蛋白质的变性处理、直接通过向小鼠腹腔注射和皮肤涂抹这两种含TAT的融合蛋白及作为对照的融合蛋白GST-GFP,一定时间作用后取体内器官和皮肤做冷冻切片,荧光显微镜检测这些融合蛋白的跨膜递送情况;并对分别融合在C端或者N端的TAT介导GFP在活体动物体内和皮肤的跨膜递送作用进行对比。结果腹腔注射实验结果表明,TAT可以介导不经过蛋白质的变性处理的融合蛋白GST-TAT-GFP和GST-GFP-TAT跨膜递送进入到小鼠的心脏、肝、肾、脾和肺,甚至脑组织;其中GST-GFP-TAT跨膜递送效率比GST-TAT-GFP更高。结构模拟分析提示GST-GFP-TAT与GST-TAT-GFP中的TAT的暴露情况不同可能是造成两种蛋白跨膜递送活性差异的重要因素。皮肤实验的结果则表明TAT不仅介导融合蛋白GST-TAT-GFP和GST-GFP-TAT进入小鼠表皮,而且使其进入小鼠皮肤的真皮层。结论 TAT可以跨膜递送不经过变性处理的融合蛋白进入小鼠皮肤和体内,递送效率可能与TAT的暴露程度相关;这些结果为在蛋白质疗法方面应用TAT提供了进一步的理论依据。     

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。     

Tat蛋白转导区域位于融合蛋白C端时的跨膜递送作用

对Tat蛋白转导区域(PTDTat)的C端融合蛋白的跨膜递送作用进行探讨.采用DNA重组技术将PTDTat融合在绿色荧光蛋白(GFP)的羧基(C)端,构建重组表达质粒pGEXGFPTat,IPTG诱导其表达后,采用谷胱甘肽Sepharose4B(GS4B)亲和层析,获得纯化的谷胱甘肽S转移酶(GST)融合的重组蛋白GSTGFPTat.该蛋白与HeLa细胞共培养,荧光显微镜观察其荧光强度随着蛋白浓度的增高而增强.流式细胞仪分析发现,在4.0μmolL蛋白浓度下,转导效率高达80.0%;而在GFP氨基(N)端含有PTDTat的融合蛋白GSTTatGFP的阳性对照组,转导效率只有32.9%.实验结果表明,C端融合的PTDTat同样具有对外源蛋白的跨膜递送作用.该结果可能为PTDTat在蛋白药物递送方面的有效应用提供理论依据.     

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。     

多聚精氨酸融合增强型绿色荧光蛋白制备方法及穿膜效果

为了方便细胞穿膜肽R9融合蛋白的可溶性表达及功能上的研究,构建了pSUMO (小分子泛素样修饰蛋白) -R9-EGFP (增强型绿色荧光蛋白) 原核表达载体。分别纯化EGFP及R9-EGFP蛋白后,作用于HepG2,细胞经流式细胞仪及激光共聚焦检测R9细胞穿膜肽的作用效果。实验结果显示在SUMO分子伴侣的作用下,R9-EGFP融合蛋白获得可溶性表达。经流式细胞仪检测,R9细胞穿膜肽可以快速有效的携带目的蛋白进入细胞内部且呈时间、剂量依赖性,大约1.5 h以后荧光强度进入平台期。共聚焦显微镜检测结果表明R9细胞穿膜肽可以有效携带EGFP进入HepG2细胞,并显示主要聚集在细胞浆内。同时体外经肝素抑制实验显示,肝素抑制R9-EGFP穿膜的效率达到50％。这些结果表明,可以利用pSUMO-R9/Ni-NTA表达纯化系统,快速、有效地表达出可溶性多聚精氨酸融合蛋白,同时R9细胞穿膜肽可以有效地携带目的蛋白进入细胞内,为进一步研究多聚精氨酸的穿膜机制提供了基础。     

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 .     

穿膜肽介导的新型基因转运载体的功能研究

目的：借助穿膜肽TAT高效跨膜的特性和LacI前头肽突变体（LacI HPM）高亲和力结合DNA的特性,建立-种安全高效、无基因插入片段大小限制的基因转导系统。方法：在TAT-LacI HPM片段C端和N端分别添加GST标签,构建pET-28a（＋）-TAT-LacI HPM-GST和pGEX-GST-TAT-LacI HPM重组表达载体,可溶性表达TAT-LacI HPM-GST及GST-TAT-LacI HPM融合蛋白并纯化,获得TAT-LacI HPM二聚体,免疫荧光检测TAT-LacI HPM融合蛋白穿过HeLa细胞膜的情况,观察EGFP的表达,用免疫印迹检测TAT-LacI HPM融合蛋白介导质粒DNA进入细胞的能力。结果：表达、纯化并获得二聚体融合蛋白,体内实验表明其具有跨膜能力,能介导带有LacI结合序列的DNA质粒进入细胞,并在转染细胞里检测到了目的蛋白。结论：初步证实TAT-LacI HPM融合蛋白作为-种新型通用性非病毒DNA转运载体的可行性,为评价这种新型DNA疫苗载体在提高免疫效果方面的可行性奠定了前期实验基础。     

结构域B在鼠冠状病毒S蛋白的抗原性及膜融合中的作用

棘突(spike, S)蛋白是冠状病毒表面必不可少的跨膜糖蛋白,在病毒进入宿主细胞时具有结合受体和诱导膜融合的双重作用。大部分冠状病毒S蛋白的受体结合域位于S1-CTD(即相对应的结构域B),而经典的乙型冠状病毒模型鼠肝炎病毒(mouse hepatitis virus, MHV)的受体mCEACAM1a与S1-NTD(即相对应的结构域A)结合,其结构域B的作用仍未完全清楚。本研究通过构建结构域B和S2膜融合元件的缺失突变体,并使其在鼠神经母细胞瘤细胞系Neuro-2a内成功表达,证实了结构域B对病毒S蛋白导致的细胞-细胞间膜融合是必需的。用不同方法处理的病毒颗粒作为抗原免疫小鼠,所获得的多克隆抗体进一步显示,结构域B不但是S蛋白的主要抗原决定簇,而且能诱导中和抗体明显抑制病毒感染和S蛋白介导的膜融合作用。此结果为阐述不同冠状病毒的致病性与感染性差异提供了新思路。     

9R穿膜肽可增强P53抗肿瘤效果

为解决P53蛋白难以进入细胞内部发挥治疗作用的瓶颈难题.将p53基因融合插入带有9个精氨酸作为穿膜肽的表达载体中表达融合蛋白CPPs-P53,并与没有穿膜肽的P53蛋白进行比较,利用Western blotting方法检测蛋白的表达情况,MTT及Annexin V/PI双染法检测细胞生长抑制率及细胞凋亡率.Western blotting检测表明已成功在原核表达系统中表达融合蛋白CPPs-P53和P53蛋白,且蛋白纯度均已达到90％以上;MTT检测表明,P53蛋白对肿瘤细胞的生长虽有一定的抑制作用,但融合蛋白CPPs-P53与之相比,对肿瘤细胞生长的抑制效果显著增强,细胞生长抑制率有明显的提升,并且细胞生长抑制率呈现剂量依赖性;Annexin V/PI双染检测细胞凋亡情况也表明P53虽可以在一定程度上诱导肿瘤细胞的凋亡,但与P53蛋白相比较,融合蛋白CPPs-P53诱导的凋亡细胞明显增加,凋亡率是P53蛋白的2～3倍.由此说明在抑制肿瘤细胞的生长和诱导细胞凋亡方面,CPPs-P53比没有穿膜肽的P53蛋白的效果更显著.     

基质金属蛋白酶-2/9敏感型可跨膜融合抗氧化酶的表达、纯化和表征北大核心CSCD

融合了跨膜肽的抗氧化酶可进入细胞,保护细胞免受放射损伤。然而跨膜肽的跨膜能力没有靶向性,其也可把抗氧化酶带入肿瘤细胞进而保护肿瘤细胞,降低放疗的效果。为此,根据多数肿瘤细胞微环境中存在活性基质金属蛋白酶(matrix metalloproteinase,MMP)-2或MMP-9的特点,在细胞跨膜肽R9与人铜、锌超氧化物歧化酶(superoxide dismutase 1,SOD1)和谷胱甘肽S-转移酶(glutathione S-transferase,GST)之间融合MMP-2/9的底物肽X,设计了融合蛋白GST-SOD1-X-R9。该蛋白在肿瘤微环境中可因MMP-2/9酶切底物肽X而失去跨膜肽,从而无法进入肿瘤细胞,进而只能进入正常细胞。全基因合成SOD1-X-R9序列,并将其插入原核表达载体pGEX-4T-1中,得到表达质粒,并实现了GST-SOD1-X-R9融合蛋白的可溶表达。GST-SOD1-X-R9经硫酸铵沉淀和GST亲和层析纯化,分子量约为47 kDa,与理论值一致。纯化的融合蛋白的SOD活性和GST活性分别为2954 U/mg和328 U/mg。GST-SOD1-X-R9的SOD活性或GST活性在生理条件下几乎没有变化。该融合蛋白在溶液中可被胶原酶Ⅳ部分水解。分别建立了2D和3D培养的HepG2细胞模型来检验肿瘤微环境中的MMP-2活力对该蛋白跨膜能力的影响。在2D培养模型中,HepG2的MMP-2活力极低,但在3D培养模型中,随着培养时间的增加,HepG2肿瘤球的体积变大,其胞外MMP-2活力也随之增强。GST-SOD1-X-R9在2D培养的HepG2细胞中具有和GST-SOD1-R9蛋白一样的跨膜效率,但在3D培养的HepG2细胞球中的跨膜能力大大降低。本研究为后续深入研究GST-SOD1-X-R9靶向防护正常细胞的氧化损伤效应奠定了基础。     

TAT-mediated endocytotic delivery of the loop deletion Bcl-2 protein protects neurons against cell death

Protein delivery mediated by protein transduction domains (PTD) such as the HIV-1 TAT-PTD has emerged as a promising approach for neuroprotection. The objective of this study was to generate and evaluate the neuroprotective potential of TAT fusion proteins using constructs based on Bcl-2 anti-death family proteins. A TAT-Bcl-2 construct with the loop domain deleted (TAT-Bcl-2Deltaloop) was tested for its ability to transduce neuronal cells and to promote survival. The potential mechanism of TAT-mediated protein internalization in neural cells was also investigated. The purified TAT-Bcl-2Deltaloop binds to neural cell and rat brain mitochondria, and transduces cultured neural cell lines and primary cortical neurons when used at nm concentrations. Effective internalization of TAT-Bcl-2Deltaloop occurs at 37 degrees C but not at 4 degrees C, consistent with an endocytotic process. Both cell association and internalization require interaction of TAT-Bcl-2Deltaloop with cell surface heparan sulfate proteoglycans. TAT-mediated protein delivery in neuronal cells occurs through a lipid raft-dependent endocytotic process, inhibited by the cholesterol-sequestering agent nystatin. Transducible loop deleted Bcl-2 increases the survival of cortical neurons following trophic factor withdrawal and also rescues neural cell lines from staurosporine-induced death. These results support the concept of using protein transduction of Bcl-2 constructs for neuroprotection.     

近红外荧光蛋白标记乳腺癌细胞外泌体的构建及鉴定

外泌体(Exosomes)是一种大小为30~100 nm的细胞外膜囊泡,与细胞的生物学功能及细胞间的信号传递有着密切的关系,尤其在癌症的诊断及治疗等领域发挥重要作用。为将外泌体更好地应用于乳腺癌肿瘤传递机制的研究,本文首先通过分子克隆手段将近红外荧光蛋白iRFP682基因和外泌体标记蛋白CD63基因克隆到含腺相关病毒(Adeno-associated virus,AAV)末端倒置重复序列(Inverted repeat terminal,ITR)的质粒载体上,构建融合表达近红外荧光蛋白和CD63蛋白的重组真核表达载体。然后再与辅助质粒共转染AAV-293细胞,包装重组腺相关病毒、纯化测量滴度后用于感染乳腺癌细胞,最后通过荧光筛选出稳定表达近红外荧光蛋白的乳腺癌细胞株。通过对乳腺癌稳定株的分离、纯化及鉴定,最终得到一个新型生物标记物：iRFP682标记的乳腺癌细胞来源的外泌体,为后续研究外泌体在乳腺癌肿瘤微环境中的分布及信号传递提供保障。     

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.     

Protein transduction as a strategy for evaluating important factors in mammalian sex determination and differentiation

Protein transduction is a powerful tool to deliver biologically active protein into mammalian cells and whole animals. Transduced proteins are folded properly and can mediate their respective functions in their hosts. To examine the feasibility of applying this strategy to study the molecular events of gonadogenesis, we have studied the kinetics of protein transduction and stability of transduced protein in in vitro mouse gonad culture systems using two reporter proteins, TAT-beta-gal and beta-gal fusion proteins with and without the TAT protein transduction domain (PTD) respectively. Our results indicate that the TAT-PTD was critical and essential for protein transduction to cultured fetal gonads. The TAT-beta-gal reporter entered the cells of the gonads and mesonephros efficiently for both sexes at E11.5 to E15.5 stages examined. The delivered protein persisted in the gonads for an extended period after an initial one-hour transduction. The distribution of the reporter was relatively even in gonads and mesonephros at E11.5 stage for both sexes and at later stages in female. The transduced protein was distributed heterogeneously in male gonads after seminiferous tubule differentiation in which the amount of reporter protein was higher outside than inside the tubules. Nevertheless, we surmise that such protein delivery technique should be useful in studies designed to evaluate the sex determining or differentiating functions of various new protein factors identified by advanced differential screening strategies.     

Revised Role of Glycosaminoglycans in TAT Protein Transduction Domain-mediated Cellular Transduction

Cellular uptake of the human immunodeficiency virus TAT protein transduction domain (PTD), or cell-penetrating peptide, has previously been surmised to occur in a manner dependent on the presence of heparan sulfate proteoglycans that are expressed ubiquitously on the cell surface. These acidic polysaccharides form a large pool of negative charge on the cell surface that TAT PTD binds avidly. Additionally, sulfated glycans have been proposed to aid in the interaction of TAT PTD and other arginine-rich PTDs with the cell membrane, perhaps aiding their translocation across the membrane. Surprisingly, however, TAT PTD-mediated induction of macropinocytosis and cellular transduction occurs in the absence of heparan sulfate and sialic acid. Using labeled TAT PTD peptides and fusion proteins, in addition to TAT PTD-Cre recombination-based phenotypic assays, we show that transduction occurs efficiently in mutant Chinese hamster ovary cell lines deficient in glycosaminoglycans and sialic acids. Similar results were obtained in cells where glycans were enzymatically removed. In contrast, enzymatic removal of proteins from the cell surface completely ablated TAT PTD-mediated transduction. Our findings support the hypothesis that acidic glycans form a pool of charge that TAT PTD binds on the cell surface, but this binding is independent of the PTD-mediated transduction mechanism and the induction of macropinocytotic uptake by TAT PTD.     

A peptide carrier for the delivery of biologically active proteins into mammalian cells.

The development of peptide drugs and therapeutic proteins is limited by the poor permeability and the selectivity of the cell membrane. There is a growing effort to circumvent these problems by designing strategies to deliver full-length proteins into a large number of cells. A series of small protein domains, termed protein transduction domains (PTDs), have been shown to cross biological membranes efficiently and independently of transporters or specific receptors, and to promote the delivery of peptides and proteins into cells. TAT protein from human immunodeficiency virus (HIV-1) is able to deliver biologically active proteins in vivo and has been shown to be of considerable interest for protein therapeutics. Similarly, the third alpha-helix of Antennapedia homeodomain, and VP22 protein from herpes simplex virus promote the delivery of covalently linked peptides or proteins into cells. However, these PTD vectors display a certain number of limitations in that they all require crosslinking to the target peptide or protein. Moreover, protein transduction using PTD-TAT fusion protein systems may require denaturation of the protein before delivery to increase the accessibility of the TAT-PTD domain. This requirement introduces an additional delay between the time of delivery and intracellular activation of the protein. In this report, we propose a new strategy for protein delivery based on a short amphipathic peptide carrier, Pep-1. This peptide carrier is able to efficiently deliver a variety of peptides and proteins into several cell lines in a fully biologically active form, without the need for prior chemical covalent coupling or denaturation steps. In addition, this peptide carrier presents several advantages for protein therapy, including stability in physiological buffer, lack of toxicity, and lack of sensitivity to serum. Pep-1 technology should be extremely useful for targeting specific protein-protein interactions in living cells and for screening novel therapeutic proteins.     

A fluorescence spectroscopy study on the interactions of the TAT-PTD peptide with model lipid membranes

Protein-transduction domains (PTDs) have been shown to translocate into and through the living cells in a rapid manner by an as yet unknown mechanism. Regardless of the mechanism of translocation, the first necessary step must be binding of the PTD peptide to the surface of the lipid membrane. We used fluorescence spectroscopy to study the interaction between PTD of the HIV-1 Tat protein (TAT-PTD; residues 47-60 of Tat, fluorescently labeled with tryptophan) and the lipid bilayer labeled with various fluorescence membrane probes. The TAT-PTD tryptophan exhibited a decrease in fluorescence intensity and an increase in anisotropy upon interaction with lipid bilayers. The fluorescence changes were linearly proportional to the density of negative charge in the membrane. Kinetic analysis of the interaction showed two apparent dissociation constants. The value of one dissociation constant (Kd1 = 2.6 +/- 0.6 microM), which accounted for 24% of the interaction, was found to be independent of the negative charge density, suggesting its nonelectrostatic nature. The value of the second dissociation constant (Kd2), which accounted for 76% of the interaction, decreased linearly from 610 +/- 150 to 130 +/- 30 microM with an increase in negative charge density from 0 to 25 mol %, suggesting this interaction is electrostatic in nature. Even though the binding was predominantly electrostatic, it could not be reversed by high salt, indicating the presence of a second, irreversible, step in the interaction with lipid. When TAT-PTD was bound to lipid vesicles labeled with 1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene (TMA-DPH), fluorescence resonance energy transfer between the tryptophan and the probe occurred at a distance of 3.4 nm. No change in fluorescence anisotropy of either TMA-DPH or DPH was observed upon the interaction with TAT-PTD, indicating no significant disruption or perturbation of the lipid bilayer by the peptide. TAT-PTD did not cause dissipation of membrane potential (165 mV, negative inside). Inclusion of 3% pyrene-labeled phosphatidylglycerol (pyrene-PG) in the membrane revealed that TAT-PTD preferentially bound to the membrane in the liquid state. We conclude that membrane fluidity is an important physicochemical parameter, which may regulate binding of TAT-PTD to the membrane.     

An experimental study on photosensitization of human liver cancer cells with sulfonated aluminum phthalocyanine (ALSPC)

The photosensitizing effect of sulfonated aluminum phthalocyanine to human liver cancer cells was studied by determining the kinetics of its cellular uptake, its state of aggregation therein and its photocytotoxicitic effect on these cells. Fluorescence methods were used to measure the cellular uptake in cell extracts and intact cells in monolayer. Results exhibited that the cellular uptake increases with the incubation time of ALSPC and saturates at 24 hrs. This relation was in coincidence with that between ALSPC's photocytotoxic effect and the ALSPC incubation time. Although the average ALSPC concentration in cells is higher than the incubation concentration of ALSPC, laser fluorescence, experiments showed that the fluorescence peak of ALSPC in cells incubated in higher concentration coincides with that of its aqueous solution of low concentration, suggesting that ALSPC in cancer cells exists in monomer state. Characteristics of the photodamaging effect of ALSPC to cancer cells were studied by both the method of typen blue staining and that of isotope labeling, which showed similar results. Results of the isotope labeling experiments implies that the DNA is damaged in ALSPC photosensitization. The photodamage on cells was also studied with 3 T 3 mouse cells (conversion), showing agreeable results to that with liver cancer cells, which suggests that ALSPC's photocytotoxic effect is nonselective to cell types.