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Cys-TAT(47-57) (Cys-[HIV-Tat (47-57)]) 是能穿透细胞的富含精氨酸的十二肽，是 HIV-1 反式激活蛋白衍生物。
Cys-TAT(47-57) (Cys-[HIV-Tat (47-57)]) is an arginine rich cell penetrating peptide derived from the HIV-1 transactivating protein.

细胞穿膜肽-说明
      穿透细胞膜进入细胞内是许多作用靶点在细胞内的生物大分子发挥作用的先决条件，然而生物膜的生物屏障作用阻止了许多高分子物质进入细胞内，从而很大程度地限制了这些物质在治疗领域的应用。因此，如何引导这些物质穿透细胞膜是一个迫切需要解决的问题，目前介导生物大分子穿透细胞膜的方法主要包括细胞穿透肽（cell penetrating peptides，CPPs）、脂质体、腺病毒、纳米颗粒、影细胞等，而CPPs是一类以非受体依赖方式，非经典内吞方式直接穿过细胞膜进入细胞的多肽，它们的长度一般不超过30个氨基酸且富含碱性氨基酸，氨基酸序列通常带正电荷。
      1型人免疫缺陷病毒转录激活因子TAT（human immunodeficiency virus-1 transcription activator， HIV-1 TAT）是第一个被发现的细胞穿透肽，它凭借一种无毒的、高效的方式进入细胞。
      细胞穿透肽（cell penetrating peptides，CPPs）的一个重要特点是可以携带多种不同大小和性质的生物活性物质进入细胞，包括小分子化合物、染料、多肽、多肽核酸（peptide nucleo acid, PNA）、蛋白质、质粒DNA、siRNA、200nm的脂质体、噬菌体颗粒和超顺磁性粒子等，这一性质为其成为靶向药物的良好载体提供了可能。
      CPPs作为载体的优势在于低毒性和无细胞类型的限制，尽管CPPs可输送不同类型的物质进入细胞，但其实际应用多集中于寡肽、蛋白质、寡聚核苷（oligonucleotides，ONs)或类似物的细胞转运。

跨膜机理
不同的细胞穿透肽（CPP）跨膜机制不同，一个细胞穿透肽（CPP）的具体机制有赖于几个参数，如分子大小（携带物质）、温度、细胞类型和细胞内外的稳定性等。细胞穿透肽（CPP）进入细胞的具体机制目前还不清楚，比较流行的推测包括以下三种：
A: 倒置胶粒模型（inverted micelle model），CPPs通过细胞膜上磷脂分子的移动形成倒置胶粒结构，而进入胞浆。
B: 直接穿透，即孔隙结构模型 (pore formation model)，CPPs在细胞膜上组成跨膜的孔隙结构而进入胞浆 。
C: 内吞方式进行细胞摄取。
来源: Cell-penetrating peptides and their therapeutic applications, Victoria Sebbage, BioscienceHorizons, Volume 2, Number 1, March 2009.

细胞穿透肽 HIV TAT
      细胞穿透肽（如HIV TAT）可以以直接穿透和内吞两种方式进入细胞。HIV TAT或者简单的多聚精氨酸可被设计作为有效的药物载体，但CPP（如HIV TAT）是如何实现胞膜转运，目前仍不清楚。
简单的HIV TAT是如何促进象直接穿透和内吞作用的入胞机制的呢？来自Gerard Wong实验室的研究人员研究了在不同的条件下，HIV TAT是如何与细胞质膜、细胞骨架、特异的胞膜受体相互作用，从而诱导了多重转运途径。

      有趣的是，TAT在不同条件下可与同一序列发生多种不同的反应，因而与胞膜、细胞骨架、特异受体相互作用可产生多种转运途径。
      CPP的跨膜机制与多肽序列存在很敏感的关系，如果在一个纯亲水性的CPP中增加一个疏水残基，就能彻底地改变其转运机制，例如，最简单的CPP原型-多聚精氨基（polyR），可以诱导细胞膜上形成跨膜的孔隙结构。疏水氨基酸通过插入胞膜来形成正曲率，精氨酸可同时形成正曲率和负曲率，赖氨酸只能沿一个方向形成负曲率，这就意味着在精氨酸与赖氨酸/疏水物之间存在补偿关系。
      如果疏水性有助于形成负高斯曲率（Gaussian curvature），那为什么TAT肽中的疏水含量相对较低呢？其原因是CPPs都是利用尽可能少的疏水基去形成saddle-splay curvature。序列上的差异很可能只会在膜上诱导短暂的类似孔隙的跨膜结构，从而形成对CPP来说更短的孔隙寿命。由于CPP的氨基酸组成不同，TAT肽在有或无受体情况下都可以介导细胞内吞作用。

     专肽生物提供各类细胞穿膜肽序列，部分由现货，例如TAT，R8，R4等，具体可咨询销售人员。

Definition

Cell permeable peptides (CPPs) are carriers with small peptide domains that can freely cross cell membranes.  They are mainly used as carriers of proteins and nucleic acids into the cell1.

Discovery

The first CPP was discovered independently by two laboratories in 1988 when it was found that the trans-activating transcriptional activator (Tat) from Human Immunodeficiency Virus 1 (HIV-1) could be efficiently taken up from the surrounding media by numerous cell types in culture2. 

Structural Characteristics

CPPs typically have an amino acid composition containing either a high relative abundance of positively charged, cationic amino acids such as lysine or arginine, or have sequences that contain an alternating pattern of polar/charged amino acids and non-polar, hydrophobic amino acids3.  Some examples include: TAT peptide-YGRKKRRQRRR, lipid membrane translocating peptide-KKAAAVLLPVLLAAP and Antennapedia leader peptide-KKWKMRRNQFWVKVQRG.

Classification

Numerous CPPs have been identified to date and they belong to a wide variety of protein families. For example, some CPPs are amphipathic protein family members3.

Mode of action

CPPs enter the cell with their carrier by either of three mechanisms:  Direct delivery that involves energy independent entry of the CPPs in to the cell4, endocytosis where the cells take up the CPPs by imbibing them with their cell membranes5 and translocation through the formation of transient structures which is yet to be understood6. 

Functions

CPPs have found numerous applications in medicine as drug delivery agents in the treatment of different diseases including cancer, virus inhibitors, contrast agents for cell labeling a classical example is Green Fluorescent protein GFP, as MRI contrast agents, quantum dots7.  TAT is very effective in delivering drugs in vitro and in vivo and so far a peptide that matches its efficiency has not been found7.

References

1.     Wagstaff KM and David JA (2006). Protein Transduction: Cell Penetrating Peptides and Their Therapeutic Applications, Current Medicinal Chemistry, 13 (12), 1371-1387.

2.     Feng S and Holland EC (1988). HIV-1 Tat trans-activation requires the loop sequence within Tar. Nature 334, 165–167.

3.     Stewart KM, Horton KL, Kelley SO (2008). Cell-penetrating peptides as delivery vehicles for biology and medicine, Org Biomol Chem., 6(13), 2242-55.

4.     Luo D, Saltzman WM (2000). Synthetic DNA delivery systems. Nat. Biotechnol, 18, 33-37.

5.     Lundberg M., Wikstrom S and Johansson M (2003). Cell surface adherence and endocytosis of protein transduction domains, Mol. Ther., 8, 143–150.

6.     Deshayes S, Gerbal-Chaloin S, Morris MC, Aldrian-Herrada G, Charnet P, Divita G (2004). On the mechanism of non-endosomial peptide-mediated cellular delivery of nucleic acids, Biochim. Biophys. Acta, 1667, 141–147.

7.     Temsamani J and Vida P (2004). The use of cell-penetrating peptides for drug delivery, Drug Discovery Today, 9 (23), 1012-1019.

Definition
Human immunodeficiency virus (HIV) is a lentivirus that causes acquired immunodeficiency syndrome (AIDS), a condition in humans in which the immune system begins to fail, leading to life-threatening opportunistic infections. Over 5000 HIV-related peptides have been synthesized, that inhibit different stages of viral life cycle.

Discovery
In 1983, two separate researchers Robert Gallo and Luc Montagnier independently declared that a novel retrovirus infecting AIDS patients. Several HIV related peptides including peptides (15-mers or 20-mers) of HIV glycoprotein 160 (gp160), gp120W16D, MN envelope (env) consensus B tat, consensus B VIF, HXB2 gag, SIVmac239, SIVmac239env, SIVmac239 gag have been used to study HIV life cycle. C34 peptide of Gp41 HIV Fragment is known as HR2, belongs to the helical region of gp41 of HIV, C-terminal heptad repeat 2 (HR2) defined as C helix or C peptide. It is known that HIV-1 enters cells by membrane fusion, C34 gp41 peptide is a potent inhibitors of HIV-1 fusion 1,2. The 86 amino acid trans-activator (Tat) protein of human immunodeficiency virus type 1 (HIV-1) is an RNA-binding transcriptional regulator. HIV-1 Tat proteins (wild type and Thr40Lys mutant) and the HIV-1 Tat peptide fragments Tat(32–48) and Tat(32–72) were chemically synthesized and used for HIV studies 3.

HIV (gp120) fragment (254-274), this fragment with sequence homology to a domain of the external envelope glycoprotein (gp120) of the human immunodeficiency virus (HIV) is important for HIV infectivity and antibody neutralization 4. HIV (gp120) fragment (421-438), derived from the CD4 attachment region of HIV gp120, inhibited the syncytial formation in vitro 5. HIV-1 gag protein p17 (76-84), HLA-A*0201-restricted immunodominant CD8 epitope of the HIV gag protein used for the characterization of CD8+ -T cells of HIV-positiv patients 6. HIV-1 rev protein (34-50), this arginine-rich fragment interacts specifically with RNA. It has been shown that rev protein and rev protein (34-50) bind IIB RNA with a similar dissociation constant of approx. 10 nM 7.

Structural Characteristics
The HIV type-1 belongs to the family Retroviridae and consists of two basic components: a core of ribonucleic acid (RNA), called the genome, and a protein component that surrounds the genome, called a capsid. The single-stranded RNA is tightly bound to nucleocapsid proteins and enzymes needed for the morphogenesis of the virion such as reverse transcriptase, proteases, ribonuclease and integrase. A matrix composed of the viral protein that surrounds the capsid. Viral envelope is composed of two layers of fatty molecules taken from the membrane of a human cell during budding process. There are 70 copies of a complex HIV protein that protrudes through the surface of the virus particle, known as Env, consists of a cap, glycoprotein (gp) 120, and a stem, gp41 molecules. This glycoprotein complex is important for fusion of virus to host cell. Both these surface proteins are important targets for treatments or HIV vaccines 8.

Mode of Action
HIV binds to a CD4 receptor and one of two co-receptors on the surface of a CD4+ T- lymphocyte. After fusion, the virus releases RNA, its genetic material, into the host cell. An HIV enzyme called reverse transcriptase converts the single- stranded HIV RNA to double-stranded HIV DNA. The newly formed HIV DNA enters the host cell's nucleus. The integrated HIV DNA is called provirus. The provirus may remain inactive for several years, producing few or no new copies of HIV. When the host cell receives a signal to become active, the provirus uses a host enzyme called RNA polymerase to create copies of the HIV genomic material, as well as shorter strands of RNA called messenger RNA (mRNA). The mRNA is used as a blueprint to make long chains of HIV proteins. An HIV enzyme called protease cuts the long chains of HIV proteins into smaller individual proteins. As the smaller HIV proteins come together with copies of HIV's RNA genetic material, a new virus particle is assembled. The newly assembled virus buds out from the host cell. During budding, the new virus acquires part of the cell's outer envelope. This envelope is embedded with viral glycoproteins which are necessary for host cell recognition.

Functions

CD8 cytotoxic, HIV-1 specific CD8 cytotoxic T lymphocyte (CTL) responses play a critical role in controlling HIV-1 replication. TCR avidity correlates with CTL function, and CTLs expressing TCRs with high avidity for their cognate MHC-viral peptide complex play an important in vivo role in neutralizing virus infections, terminating virus infection and delaying systemic AIDS virus dissemination from the mucosal inoculation site.

HIV-1 envelope transmembrane protein that contain highly positively charged amphipathic helices (designated LLP) in have both cytolytic and calmodulin (CaM) binding/inhibitory properties that contribute to cytopathogenesis during a viral infection.

HIV-1 vif, The human immunodeficiency virus type 1 (HIV-1) auxiliary gene vif is essential for virus propagation in peripheral blood lymphocytes, macrophages, and in some T-cell lines. (i) Vif protein binds HIV-1 PR (protease), but not covalently linked tethered PR-PR; (ii) the four amino acids residing at the N terminus of HIV-1 PR are essential for Vif/PR interaction; (iii) synthetic peptide derived from the N terminus of HIV-1 PR inhibits Vif/PR binding; and (iv) this peptide inhibits the propagation of HIV-1 in restrictive cells 9.

References

1.     Bianchi E, Finotto M, Ingallinella P, Hrin R, Carella AV, Hou XS, Schleif WA, Miller MD, Geleziunas R, Pessi A (2005). Covalent stabilization of coiled coils of the HIV gp41 N region yields extremely potent and broad inhibitors of viral infection. PNAS., 102(36):12903-12908

2.     de Rosny E, Vassell R, Jiang S, Kunert R, Weiss CD (2004). Binding of the 2F5 monoclonal antibody to native and fusion-intermediate forms of human immunodeficiency virus type 1 gp41: implications for fusion-inducing conformational changes. J. Virol., 78(5):2627-2631.
3.     Klostermeier D, Bayer P, Kraft M, Frank RW, Rösch P (1997). Spectroscopic investigations of HIV-1 trans-activator and related peptides in aqueous solutions. Biophysical Chemistry, 63(2):87-96.

4.     Ho DD, Kaplan JC, Rackauskas IE, Gurney ME (1988). Second conserved domain of gp120 is important for HIV infectivity and antibody neutralization. Science, 239(4843):1021-1023.

5.     Morrow WJ, Williams WM, Whalley AS, Ryskamp T, Newman R, Kang CY, Chamat S, Köhler H, Kieber-Emmons T (1992). Synthetic peptides from a conserved region of gp120 induce broadly reactive anti-HIV responses. Immunology, 75(4):557-564.

6.     Wilkinson J, Cope A, Gill J, Bourboulia D, Hayes P, Imami N, Kubo T, Marcelin A, Calvez V, Weiss R, Gazzard B, Boshoff C, Gotch F (2002). Identification of Kaposi's sarcoma-associated herpesvirus (KSHV)-specific cytotoxic T-lymphocyte epitopes and evaluation of reconstitution of KSHV-specific responses in human immunodeficiency virus type 1-Infected patients receiving highly active antiretroviral therapy. J. Virol., 76(6):2634-2640.

7.     Kjems J, Calnan BJ, Frankel AD, Sharp PA (1992). Specific binding of a basic peptide from HIV-1 Rev. EMBO J., 11(3):1119-29.
8.     Chan DC, Fass D, Berger JM, Kim PS (1997). Core structure of gp41 from the HIV   envlope glycoprotein . Cell, 89:263–73.

9.     Hutoran M, Britan E, Baraz L, Blumenzweig I, Steinitz M, Kotler M (2004). Abrogation of Vif function by peptide derived from the N-terminal region of the human immunodeficiency virus type 1 (HIV-1) protease. Virology, 330(1):261-270.

多肽H2N-Cys-Tyr-Gly-Arg-Lys-Lys-Arg-Arg-Gln-Arg-Arg-Arg-NH2的合成步骤：

1、合成MBHA树脂：取若干克的MBHA树脂（如初始取代度为0.5mmol/g）和1倍树脂摩尔量的Fmoc-Linker-OH加入到反应器中，加入DMF，搅拌使氨基酸完全溶解。再加入树脂2倍量的DIEPA，搅拌混合均匀。再加入树脂0.95倍量的HBTU，搅拌混合均匀。反应3-4小时后，用DMF洗涤3次。用2倍树脂体积的10%乙酸酐/DMF 进行封端30分钟。然后再用DMF洗涤3次，甲醇洗涤2次，DCM洗涤2次，再用甲醇洗涤2次。真空干燥12小时以上，得到干燥的树脂{Fmoc-Linker-MHBA Resin}，测定取代度。这里测得取代度为 0.3mmol/g。结构如下图：

2、脱Fmoc：取2.02g的上述树脂，用DCM或DMF溶胀20分钟。用DMF洗涤2遍。加3倍树脂体积的20%Pip/DMF溶液，鼓氮气30分钟，然后2倍树脂体积的DMF 洗涤5次。得到 H2N-Linker-MBHA Resin 。（此步骤脱除Fmoc基团，茚三酮检测为蓝色，Pip为哌啶）。结构图如下：

3、缩合：取1.82mmol Fmoc-Arg(Pbf)-OH 氨基酸，加入到上述树脂里，加适当DMF溶解氨基酸，再依次加入3.64mmol DIPEA，1.73mmol HBTU。反应30分钟后，取小样洗涤，茚三酮检测为无色。用2倍树脂体积的DMF 洗涤3次树脂。（洗涤树脂，去掉残留溶剂，为下一步反应做准备）。得到Fmoc-Arg(Pbf)-Linker-MBHA Resin。氨基酸：DIPEA：HBTU：树脂=3：6：2.85：1（摩尔比）。结构图如下：

4、依次循环步骤二、步骤三，依次得到

H2N-Arg(Pbf)-Linker-MBHA Resin

Fmoc-Arg(Pbf)-Arg(Pbf)-Linker-MBHA Resin

H2N-Arg(Pbf)-Arg(Pbf)-Linker-MBHA Resin

Fmoc-Arg(Pbf)-Arg(Pbf)-Arg(Pbf)-Linker-MBHA Resin

H2N-Arg(Pbf)-Arg(Pbf)-Arg(Pbf)-Linker-MBHA Resin

Fmoc-Gln(Trt)-Arg(Pbf)-Arg(Pbf)-Arg(Pbf)-Linker-MBHA Resin

H2N-Gln(Trt)-Arg(Pbf)-Arg(Pbf)-Arg(Pbf)-Linker-MBHA Resin

Fmoc-Arg(Pbf)-Gln(Trt)-Arg(Pbf)-Arg(Pbf)-Arg(Pbf)-Linker-MBHA Resin

H2N-Arg(Pbf)-Gln(Trt)-Arg(Pbf)-Arg(Pbf)-Arg(Pbf)-Linker-MBHA Resin

Fmoc-Arg(Pbf)-Arg(Pbf)-Gln(Trt)-Arg(Pbf)-Arg(Pbf)-Arg(Pbf)-Linker-MBHA Resin

H2N-Arg(Pbf)-Arg(Pbf)-Gln(Trt)-Arg(Pbf)-Arg(Pbf)-Arg(Pbf)-Linker-MBHA Resin

Fmoc-Lys(Boc)-Arg(Pbf)-Arg(Pbf)-Gln(Trt)-Arg(Pbf)-Arg(Pbf)-Arg(Pbf)-Linker-MBHA Resin

H2N-Lys(Boc)-Arg(Pbf)-Arg(Pbf)-Gln(Trt)-Arg(Pbf)-Arg(Pbf)-Arg(Pbf)-Linker-MBHA Resin

Fmoc-Lys(Boc)-Lys(Boc)-Arg(Pbf)-Arg(Pbf)-Gln(Trt)-Arg(Pbf)-Arg(Pbf)-Arg(Pbf)-Linker-MBHA Resin

H2N-Lys(Boc)-Lys(Boc)-Arg(Pbf)-Arg(Pbf)-Gln(Trt)-Arg(Pbf)-Arg(Pbf)-Arg(Pbf)-Linker-MBHA Resin

Fmoc-Arg(Pbf)-Lys(Boc)-Lys(Boc)-Arg(Pbf)-Arg(Pbf)-Gln(Trt)-Arg(Pbf)-Arg(Pbf)-Arg(Pbf)-Linker-MBHA Resin

H2N-Arg(Pbf)-Lys(Boc)-Lys(Boc)-Arg(Pbf)-Arg(Pbf)-Gln(Trt)-Arg(Pbf)-Arg(Pbf)-Arg(Pbf)-Linker-MBHA Resin

Fmoc-Gly-Arg(Pbf)-Lys(Boc)-Lys(Boc)-Arg(Pbf)-Arg(Pbf)-Gln(Trt)-Arg(Pbf)-Arg(Pbf)-Arg(Pbf)-Linker-MBHA Resin

H2N-Gly-Arg(Pbf)-Lys(Boc)-Lys(Boc)-Arg(Pbf)-Arg(Pbf)-Gln(Trt)-Arg(Pbf)-Arg(Pbf)-Arg(Pbf)-Linker-MBHA Resin

Fmoc-Tyr(tBu)-Gly-Arg(Pbf)-Lys(Boc)-Lys(Boc)-Arg(Pbf)-Arg(Pbf)-Gln(Trt)-Arg(Pbf)-Arg(Pbf)-Arg(Pbf)-Linker-MBHA Resin

H2N-Tyr(tBu)-Gly-Arg(Pbf)-Lys(Boc)-Lys(Boc)-Arg(Pbf)-Arg(Pbf)-Gln(Trt)-Arg(Pbf)-Arg(Pbf)-Arg(Pbf)-Linker-MBHA Resin

Fmoc-Cys(Trt)-Tyr(tBu)-Gly-Arg(Pbf)-Lys(Boc)-Lys(Boc)-Arg(Pbf)-Arg(Pbf)-Gln(Trt)-Arg(Pbf)-Arg(Pbf)-Arg(Pbf)-Linker-MBHA Resin

以上中间结构，均可在专肽生物多肽计算器-多肽结构计算器中，一键画出。

最后再经过步骤二得到 H2N-Cys(Trt)-Tyr(tBu)-Gly-Arg(Pbf)-Lys(Boc)-Lys(Boc)-Arg(Pbf)-Arg(Pbf)-Gln(Trt)-Arg(Pbf)-Arg(Pbf)-Arg(Pbf)-Linker-MBHA Resin，结构如下：

5、切割：6倍树脂体积的切割液（或每1g树脂加8ml左右的切割液），摇床摇晃 2小时，过滤掉树脂，用冰无水乙醚沉淀滤液，并用冰无水乙醚洗涤沉淀物3次，最后将沉淀物放真空干燥釜中，常温干燥24小试，得到粗品H2N-Cys-Tyr-Gly-Arg-Lys-Lys-Arg-Arg-Gln-Arg-Arg-Arg-NH2。结构图见产品结构图。

切割液选择：1）TFA：H2O=95%：5%

2）TFA：H2O：TIS=95%：2.5%：2.5%

3）三氟乙酸：茴香硫醚：1，2-乙二硫醇：苯酚：水=87.5%：5%：2.5%：2.5%：2.5%

（前两种适合没有容易氧化的氨基酸，例如Trp、Cys、Met。第三种适合几乎所有的序列。）

6、纯化冻干：使用液相色谱纯化，收集目标峰液体，进行冻干，获得蓬松的粉末状固体多肽。不过这时要取小样复测下纯度 是否目标纯度。

7、最后总结：

杭州专肽生物技术有限公司（ALLPEPTIDE https://www.allpeptide.com）主营定制多肽合成业务，提供各类长肽，短肽，环肽，提供各类修饰肽，如：荧光标记修饰（CY3、CY5、CY5.5、CY7、FAM、FITC、Rhodamine B、TAMRA等），功能基团修饰肽（叠氮、炔基、DBCO、DOTA、NOTA等），同位素标记肽（N15、C13），订书肽（Stapled Peptide）,脂肪酸修饰肽（Pal、Myr、Ste），磷酸化修饰肽（P-Ser、P-Thr、P-Tyr），环肽（酰胺键环肽、一对或者多对二硫键环），生物素标记肽，PEG修饰肽，甲基化修饰肽等。

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