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| Targets |
SV40 large T antigen NLS specifically targets importin-alpha (karyopherin-alpha), an adaptor protein that recognizes and binds to classical NLSs. The binding is mediated by the positively charged residues of the NLS (basic amino acids lysine and arginine) interacting with the acidic residues in the major and minor NLS-binding pockets of importin-alpha. Structural studies (e.g., PDB 1ejl) have shown that the monopartite SV40 large T antigen NLS binds to two binding sites on importin-alpha, similar to what is observed in yeast importin-alpha. After binding to importin-alpha, the NLS-cargo complex interacts with importin-beta, which facilitates translocation through the nuclear pore complex into the nucleus. The SV40 NLS is not an inhibitor; it is a substrate of the nuclear import machinery.
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| ln Vitro |
The nuclear localization sequence (NLS) is flanked by the protein kinase CK2 (CK2) site (Ser111Ser112), which enhances the nuclear import of SV40 big tumor-antigen (T-ag)[1]. Although it has been demonstrated that PK-C inhibits the nuclear import of certain proteins, including lamin B2, T-ag NLS can increase nuclear import[2].
In vitro, SV40 large T antigen NLS has been extensively used to study the nuclear import mechanism. In a typical cell-free import assay, fluorescently labeled (e.g., FITC-conjugated) SV40 NLS is mixed with importin-alpha and importin-beta and incubated with permeabilized cells or isolated nuclei. The NLS peptide is imported into the nucleus in an energy-dependent manner, demonstrating its function as a bona fide NLS. The binding affinity between SV40 NLS and importin-alpha has been measured by surface plasmon resonance (SPR) or isothermal titration calorimetry (ITC), with KD values typically in the micromolar range (e.g., 5-20 microM). When conjugated to non-nuclear proteins (e.g., BSA, GFP) or nanoparticles, SV40 NLS can redirect these cargos to the nucleus, confirming its ability to function as a transport signal. |
| ln Vivo |
In vivo, SV40 large T antigen NLS has been used to study nuclear import in living cells and animals. When microinjected into cells or expressed as a fusion with heterologous proteins, the SV40 NLS directs the cargo to the nucleus. Studies using fluorescently labeled SV40 NLS in living cells have provided insights into the dynamics of nuclear import, including the roles of importins, the nuclear pore complex, and Ran GTPase. In animal models, SV40 NLS has been used to enhance the delivery of genes, proteins, and nanoparticles to the nucleus. For example, attaching SV40 NLS to non-viral gene delivery vectors (e.g., polyethylenimine, liposomes) can significantly increase transfection efficiency by facilitating the nuclear import of plasmid DNA.
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| Enzyme Assay |
A typical non-cellular (cell-free) protocol for evaluating the binding of SV40 large T antigen NLS to importin-alpha uses surface plasmon resonance (SPR). Importin-alpha is immobilized on a CM5 sensor chip via amine coupling according to the manufacturer's instructions. The SV40 NLS peptide is synthesized with a biotin tag at the N-terminus or used as a synthetic peptide. Various concentrations of SV40 NLS (0.1-100 microM) are injected over the sensor chip in running buffer (10 mM HEPES pH 7.4, 150 mM NaCl, 0.005% Tween-20) at a flow rate of 30 microL/min for 60 seconds, followed by 120 seconds dissociation. The chip is regenerated with a 30-second pulse of 10 mM glycine-HCl (pH 2.0) after each cycle. The binding affinity (KD) is calculated by fitting the sensorygrams to a 1:1 Langmuir binding model. Alternatively, a competitive binding assay can be performed by incubating importin-alpha with a fixed concentration of a known NLS (e.g., FITC-labeled SV40 NLS) and increasing concentrations of unlabeled SV40 NLS, followed by fluorescence polarization or anisotropy measurement.
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| Cell Assay |
A typical in vitro cellular protocol for evaluating the nuclear import activity of SV40 large T antigen NLS uses digitonin-permeabilized cells (e.g., HeLa cells). HeLa cells are grown on coverslips to 50-70% confluence. The cells are washed with transport buffer (TB: 20 mM HEPES pH 7.3, 110 mM potassium acetate, 2 mM magnesium acetate, 1 mM EGTA, 2 mM DTT) and permeabilized with 40 microg/mL digitonin in TB for 5 minutes on ice. The permeabilized cells are washed with TB and incubated with an import mixture containing 2 microM FITC-labeled SV40 NLS peptide, 2 microM importin-alpha, 2 microM importin-beta, 2 mM ATP, and an ATP-regenerating system (0.5 mM creatine phosphate, 10 microg/mL creatine kinase) in TB for 20-60 minutes at 30degC. Negative control reactions omit ATP or importins, or use a mutant NLS (e.g., PKKKRKV → PKKTKRKV). After incubation, cells are washed, fixed with 4% paraformaldehyde, stained with DAPI, and examined by fluorescence microscopy. Nuclear import is quantified by measuring the ratio of nuclear fluorescence to cytoplasmic fluorescence using image analysis software.
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| Animal Protocol |
An in vivo animal protocol for evaluating SV40 NLS-enhanced gene delivery uses a mouse model of a target disease (e.g., cancer). Polyethylenimine (PEI)-based nanoparticles are complexed with plasmid DNA (e.g., encoding luciferase or a therapeutic gene) either with or without SV40 NLS conjugated to the nanoparticle surface or directly conjugated to the DNA via a DNA-binding domain. The nanoparticles are administered intravenously (tail vein) or intratumorally to mice bearing subcutaneous tumor xenografts. At various time points post-administration (e.g., 6, 12, 24, 48, 72 hours), mice are euthanized, and organs (tumor, liver, spleen, kidney, lung) are harvested. The expression of the reporter gene (e.g., luciferase) is measured by a bioluminescence assay or by measuring enzyme activity in tissue lysates. Gene delivery efficiency is compared between SV40 NLS-modified and unmodified nanoparticles. For imaging experiments, animals are injected with D-luciferin, and bioluminescence imaging (BLI) is performed to track gene expression in live animals.
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| ADME/Pharmacokinetics |
The pharmacokinetics of SV40 large T antigen NLS as a peptide are characterized by rapid degradation and short half-life. The peptide is composed of 7 amino acids (PKKKRKV) and has a molecular weight of approximately 911 Da. When administered intravenously, the peptide is rapidly cleared from the circulation with a half-life of minutes due to proteolytic degradation by serum peptidases and rapid renal clearance. For in vivo applications as a nuclear delivery enhancer, SV40 NLS is typically conjugated to larger carriers (proteins, nanoparticles, polymers) to reduce clearance and protect the peptide from degradation. When conjugated, the NLS serves as a targeting moiety rather than a standalone drug. The pharmacokinetics of the conjugated carrier are determined primarily by the carrier itself, with the NLS tag having a minimal effect on the overall PK profile.
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| Toxicity/Toxicokinetics |
Toxicity data specific to the SV40 large T antigen NLS peptide are not available as it is a research tool rather than a therapeutic agent. The peptide is derived from a viral protein but is a short synthetic peptide (7 amino acids) and is not considered toxic at the concentrations used in research (typically 1-20 microM). In cell culture, SV40 NLS has no apparent cytotoxic effects. When conjugated to gene delivery vectors, the toxicity is determined by the vector (e.g., PEI, liposomes) rather than the NLS itself. Standard laboratory safety precautions should be followed when handling the peptide, including the use of gloves, lab coats, and safety glasses. The lyophilized peptide should be stored at -20degC, protected from light and moisture. It is for research use only and should not be used in humans or animals for therapeutic or diagnostic purposes.
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| References |
[1]. Xiao CY, et al. Negative charge at the protein kinase CK2 site enhances recognition of the SV40 large T-antigen NLS by importin: effect of conformation. FEBS Lett. 1998 Dec 4;440(3):297-301.
[2]. Xiao CY, et al. An engineered site for protein kinase C flanking the SV40 large T-antigen NLS confers phorbol ester-inducible nuclear import. FEBS Lett. 1998 Oct 9;436(3):313-7. |
| Additional Infomation |
SV40 large T antigen NLS is a synthetic peptide corresponding to the nuclear localization signal of the Simian Virus 40 (SV40) large T antigen. The peptide sequence is PKKKRKV (Pro-Lys-Lys-Lys-Arg-Lys-Val). It is a classic monopartite NLS that binds to importin-alpha (karyopherin-alpha) and mediates the nuclear import of cargo proteins. SV40 NLS is a fundamental tool in cell biology research for studying nucleocytoplasmic transport, protein nuclear import, and the mechanisms of nuclear pore complex function. It is also used to enhance the nuclear delivery of non-viral gene therapy vectors. The peptide is commercially available and is typically supplied as a lyophilized powder with a purity of ≥95%. It is soluble in water and PBS. The peptide should be stored at -20degC and protected from light. It is for research use only and is not approved for clinical use.
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| Molecular Formula |
C58H104N20O18S
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| Molecular Weight |
1401.63457107544
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| Exact Mass |
1400.755
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| CAS # |
163815-24-5
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| PubChem CID |
156599041
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| Appearance |
Typically exists as solid at room temperature
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| LogP |
-14.1
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| Hydrogen Bond Donor Count |
22
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| Hydrogen Bond Acceptor Count |
25
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| Rotatable Bond Count |
51
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| Heavy Atom Count |
97
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| Complexity |
2660
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| Defined Atom Stereocenter Count |
10
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| SMILES |
CC(C)[C@@H](C(=O)N[C@@H](CCC(=O)O)C(=O)N[C@@H](CC(=O)O)C(=O)O)NC(=O)[C@H](CCCCN)NC(=O)[C@H](CCCN=C(N)N)NC(=O)[C@H](CCCCN)NC(=O)[C@H](CCCCN)NC(=O)[C@H](CCCCN)NC(=O)[C@@H]1CCCN1C(=O)CNC(=O)CNC(=O)CNC(=O)[C@H](CS)N
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| InChi Key |
BKONVPKKIKALJE-HQIXXVJESA-N
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| InChi Code |
InChI=1S/C58H104N20O18S/c1-32(2)47(56(94)75-39(19-20-45(82)83)53(91)76-40(57(95)96)27-46(84)85)77-54(92)37(16-6-10-24-62)72-52(90)38(17-11-25-66-58(64)65)73-50(88)35(14-4-8-22-60)70-49(87)34(13-3-7-21-59)71-51(89)36(15-5-9-23-61)74-55(93)41-18-12-26-78(41)44(81)30-68-42(79)28-67-43(80)29-69-48(86)33(63)31-97/h32-41,47,97H,3-31,59-63H2,1-2H3,(H,67,80)(H,68,79)(H,69,86)(H,70,87)(H,71,89)(H,72,90)(H,73,88)(H,74,93)(H,75,94)(H,76,91)(H,77,92)(H,82,83)(H,84,85)(H,95,96)(H4,64,65,66)/t33-,34-,35-,36-,37-,38-,39-,40-,41-,47-/m0/s1
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| Chemical Name |
(2S)-2-[[(2S)-2-[[(2S)-2-[[(2S)-6-amino-2-[[(2S)-2-[[(2S)-6-amino-2-[[(2S)-6-amino-2-[[(2S)-6-amino-2-[[(2S)-1-[2-[[2-[[2-[[(2R)-2-amino-3-sulfanylpropanoyl]amino]acetyl]amino]acetyl]amino]acetyl]pyrrolidine-2-carbonyl]amino]hexanoyl]amino]hexanoyl]amino]hexanoyl]amino]-5-(diaminomethylideneamino)pentanoyl]amino]hexanoyl]amino]-3-methylbutanoyl]amino]-4-carboxybutanoyl]amino]butanedioic acid
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| HS Tariff Code |
2934.99.9001
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| Storage |
Powder -20°C 3 years 4°C 2 years In solvent -80°C 6 months -20°C 1 month Note: Please store this product in a sealed and protected environment, avoid exposure to moisture. |
| Shipping Condition |
Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)
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| Solubility (In Vitro) |
May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples
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| Solubility (In Vivo) |
Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.
Injection Formulations
Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)(e.g. IP/IV/IM/SC) *Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution. Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline) Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil) Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals). View More
Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)] Oral Formulations
Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium) Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals). View More
Oral Formulation 3: Dissolved in PEG400 (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 0.7135 mL | 3.5672 mL | 7.1345 mL | |
| 5 mM | 0.1427 mL | 0.7135 mL | 1.4269 mL | |
| 10 mM | 0.0713 mL | 0.3567 mL | 0.7135 mL |
*Note: Please select an appropriate solvent for the preparation of stock solution based on your experiment needs. For most products, DMSO can be used for preparing stock solutions (e.g. 5 mM, 10 mM, or 20 mM concentration); some products with high aqueous solubility may be dissolved in water directly. Solubility information is available at the above Solubility Data section. Once the stock solution is prepared, aliquot it to routine usage volumes and store at -20°C or -80°C. Avoid repeated freeze and thaw cycles.
Calculation results
Working concentration： mg/mL；
Method for preparing DMSO stock solution： mg drug pre-dissolved in μL DMSO (stock solution concentration mg/mL). Please contact us first if the concentration exceeds the DMSO solubility of the batch of drug.
Method for preparing in vivo formulation:：Take μL DMSO stock solution, next add μL PEG300, mix and clarify, next addμL Tween 80, mix and clarify, next add μL ddH2O，mix and clarify.
(1) Please be sure that the solution is clear before the addition of next solvent. Dissolution methods like vortex, ultrasound or warming and heat may be used to aid dissolving.
(2) Be sure to add the solvent(s) in order.
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