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元件指南
VectorBuilder提供了许多常用的载体元件，可供用户在设计载体时进行选择。下表按类型提供了这些常用元件的详细信息。
表位标签蛋白
| 姓名 | 描述 | Application Notes | References | 序列 |
|---|---|---|---|---|
| FLAG | FLAG epitope tag | Facilitates antibody binding of tagged protein. | J Biochem Biophys Methods. 49:455 (2001); Appl Microbiol Biotechnol. 60:523 (2003) | View |
| 3xFLAG | 3 tandem FLAG epitope tag | Facilitates antibody binding of tagged protein. | Appl Microbiol Biotechnol. 60:523 (2003) | View |
| HA | Hemagglutinin epitope tag | Facilitates antibody binding of tagged protein. | Nature. 443:230 (2006) | View |
| Myc | c-Myc epitope tag | Facilitates antibody binding of tagged protein. | Appl Microbiol Biotechnol. 60:523 (2003) | View |
| V5 | V5 epitope tag | Facilitates antibody binding of tagged protein. | J Biol Chem. 282:20799 (2007) | View |
| OVA(257-264) | H-2Kb-restricted OVA MHC class I epitope | Allows detection of a strong CD8+ cytolytic T cell response. | Eur J Immunol. 21:2891 (1991); J Immunol. 150:1212 (1993) | View |
| OVA(323-339) | I-Ad-restricted OVA MHC class II epitope | Allows studying the nature of class II MHC-peptide binding and T-cell activation. | Biochemistry. 38:16663 (1999); J Immunol. 171:1999 (2003) | View |
| 10xGCN4_v4 | The v4 version of the GCN4 peptide array with 10 copies of the peptide binding site, also know as SunTag10x_v4 | Allows tagged proteins to be recognized by the GCN4 scFv antibody. | Cell. 159: 635 (2014) | View |
| Rho1D4 | A 9 amino acid 1D4 epitope derived from the C-terminus of bovine rhodopsin | Allows tagged proteins to be recognized by the Rho1D4 antibody; typically recommended for the purification and characterization of membrane proteins. | Methods Mol Biol. 1177:1 (2014) | View |
| ALFA-tag | ALFA-tag | Allows tagged proteins to be recognized by the NbALFA nanobody. | Nature Communications. 10:4403 (2019) | View |
纯化标签
| 姓名 | 描述 | Application Notes | References | 序列 |
|---|---|---|---|---|
| 6xHis | 6 tandem histidine tag | Facilitates purification by nickel or cobalt matrix and antibody binding of tagged protein. | Appl Microbiol Biotechnol. 60:523 (2003) | View |
| 10xHis | 10 tandem histidine tag | Facilitates purification by nickel or cobalt matrix and antibody binding of tagged protein. | Appl Microbiol Biotechnol. 60:523 (2003) | View |
| Strep-tag_II | Strep-tag II | Facilitates purification by Strep-Tactin (modified streptavidin) matrix and antibody binding of tagged protein. | Appl Microbiol Biotechnol. 60:523 (2003) | View |
| Twin-Strep-tag | 2 tandem Strep-tag II linked with 3xGGGS | Facilitates purification by Strep-Tactin (modified streptavidin) matrix and antibody binding of tagged protein. | Protein Expr Purif. 92:54 (2013) | View |
| S-tag | S-tag | Facilitates purification by S-protein matrix and antibody binding of tagged protein. | Appl Microbiol Biotechnol. 60:523 (2003) | View |
| MBP | Maltose-binding protein | Facilitates purification by amylose matrix and antibody binding of tagged protein; increases the solubility of tagged protein in bacteria. | Appl Microbiol Biotechnol. 60:523 (2003) | View |
| GST | Glutathione S-transferase | Facilitates purification by glutathione matrix and antibody binding of tagged protein; helps to stabilize tagged protein and protect it against intracellular protease cleavage. | Appl Microbiol Biotechnol. 60:523 (2003) | View |
| Avi | Avi tag | Facilitates biotinylation of tagged protein by the enzyme BirA so the protein can be isolated by streptavidin. | Protein Sci. 8:921 (1999); Methods Mol Biol. 1266: 171 (2015) | View |
| Halo | HaloTag7 | Increases expression and solubility of tagged protein; enables covalent, efficient and specific capture of a protein of interest onto HaloLink™ Resin; high yield and purity. | Protein Expr Purif. 68:110 (2009) | View |
| Halo-TEV | HaloTag7 linked with TEV site | Increases expression and solubility of tagged protein; enables covalent, efficient and specific capture of a protein of interest onto HaloLink™ Resin; high yield and purity; can be removed by TEV protease; N-terminal tag. | Protein Expr Purif. 68:110 (2009) | View |
| SUMO | Small ubiquitin-like modifier tag | Facilitates purification by enhancing stability and solubility of tagged proteins; can be specifically recognized by SUMO proteases. | Protein Sci.22(11):1466-77 (2013) | View |
| hIgG-Fc | The constant region (CH2 and CH3 domains) of human immunoglobulin heavy chain | Increases protein yield; allows rapid and simple detection of protein expression; enables simple protein G/A affinity chromatography based purification of Fc-tagged proteins. | EMBO Mol Med. 4:1015 (2012); J Biomed Res. 30: 209 (2016) | View |
| mlgG-Fc | The constant region (CH2 and CH3 domains) of mouse immunoglobulin heavy chain | Increases protein yield; allows rapid and simple detection of protein expression; enables simple protein G affinity chromatography based purification of Fc-tagged proteins. | EMBO Mol Med. 4:1015 (2012); Sci Rep. 6:30171 (2016) | View |
| NpuC(HN) | A modified C-terminal segment of the Nostoc punctiforme (Npu) DnaE split intein | Facilitates purification of N-terminal tagged proteins by intein-mediated cleavage and subsequent release of tagless target proteins induced by a simple pH shift (8.5 to 6.2) which leads to a cyclization reaction of the C-terminal asparagine to form a succinimide ring. | Curr Protoc Protein Sci. 91:5.29.1 (2018) | View |
蛋白酶切割位点
| 姓名 | 描述 | Application Notes | References | 序列 |
|---|---|---|---|---|
| EK | Bovine enterokinase cleavage site | Cleaved by enterokinase, which cuts after lysine in the recognition sequence DDDDK. | Appl Microbiol Biotechnol. 60:523 (2003) | View |
| TEV | Tobacco etch virus protease cleavage site | Cleaved by tobacco etch virus protease, which cuts after glutamine in the recognition sequence EXXYXQ(G/S) where X can be any amino acid. | Appl Microbiol Biotechnol. 60:523 (2003) | View |
| Thrombin | Thrombin cleavage site | Cleaved by thrombin, which cuts after the fourth residue in the recognition sequence XXP(R/K)XX where XX on the N end are hydrophobic residues and XX on the C end are non-acidic residues. | Appl Microbiol Biotechnol. 60:523 (2003) | View |
| Factor-Xa | Factor Xa cleavage site | Cleaved by factor Xa, which cuts after arginine in the recognition sequence I(E/D)GR. | Appl Microbiol Biotechnol. 60:523 (2003) | View |
| HRV3C | Human rhinovirus 3C protease site | Cleaved by human rhinovirus 3C protease, the cleavage site is between Gln and Gly in the recognition sequences LEVLFQ↓GP. | Cell.168(3):427-441 (2017) | View |
信号肽 (for directing subcellular localization of proteins, such as nuclear localization, membrane localization, secretion, etc.)
| 姓名 | 描述 | Application Notes | References | 序列 |
|---|---|---|---|---|
| SV40-NLS | Simian virus 40 nuclear localization signal | Directs nuclear localization of tagged protein. | Cell. 39:499 (1984) | View |
| 3xNLS | 3 tandem simian virus 40 nuclear localization signal | Directs nuclear localization of tagged protein. | J Immunol. 182: 3173 (2009) | View |
| cMYC-NLS | Nuclear localization signal of human c-MYC gene | Directs nuclear localization of tagged protein. | Mol Cell Biol. 8:4048 (1988);Bioconjug Chem. 26: 1004 (2015) | View |
| BPSV40-NLS | A classical nuclear localization signal (NLS) consists of two clusters of basic residues separated by 10–12 residues (bipartite), NLS-containing cargoes are imported by a heterodimeric import receptor complex composed of importin α and importin β | Directs nuclear localization of tagged protein. | J Biol Chem. 276:1317 (2001); Biophys J. 96:3840 (2009); Nature. 540:144 (2016); Nat Methods. 16:887-893 (2019) | View |
| MITO | Mitochondrial targeting sequence from human cytochrome c oxidase subunit VIII gene | Directs tagged protein to the intermembrane space of mitochondria. | Nature. 358:325 (1992); Proc Natl Acad Sci U S A. 96: 11752 (1999) | View |
| Igκ-sig | Murine Ig kappa chain leader sequence | Directs tagged protein to the secretory pathway. | J Immunol Methods. 152:89 (1992) | View |
| α-Factor | Yeast pheromone α-factor leader sequence | Directs export of tagged protein in yeast into culture medium. | Gene. 54:175 (1987) | View |
| HBM | Honeybee melittin signal peptide | Allows enhanced secretion of tagged proteins from insect cells into the medium. | Gene. 98:177 (1991) | View |
| PelB | Pectate lyase B leader sequence | Directs tagged protein to the bacterial periplasm, where the sequence is removed by a signal peptidase. | Protein Expr Purif. 54:227 (2007) | View |
| NES | Nuclear export signal | Directs nuclear export of tagged protein. | PLoS One.10:e0137652 (2015) | View |
| Lyn | N-terminal myristoylation and palmitoylation sequence from Lyn, a Src family tyrosine kinase | Anchors the tagged protein to the plasma membrane. | J Neurosci Methods. 93:37 (1999); J Cell Biol. 174:255 (2006) | View |
| SNAP25 | A protein which is synthesized on free ribosomes and recruited to the inner surface of plasma membrane after the palimitoylation of specific cystesine residues | Directs the tagged proteins to the inner surface of plasma membrane. | EMBO J. 16:1575 (1997) | View |
| GPI | Plasma membrane glycosylphosphatidylinositol anchoring signal | Anchors glycoprotein to the plasma membrane; helps increase the mobility of membrane proteins therefore supporting signal transduction and cellular transport; plays an important role in creating antigens on cell surface. | Genesis. 44:202 (2006) | View |
| TMD-ST | Transmembrane domain of rat liver sialyltransferase | Directs the tagged protein to Golgi. | J Biol Chem. 270:5483 (1995) | View |
| PDGFR | Platelet-derived growth factor receptor beta transmembrane domain | Anchors the tagged protein to the extracellular side of plasma membrane for displaying, usually works with secretory signal peptide. | Proc Natl Acad Sci U S A. 85:3435 (1988); J Biol Chem. 285:12268 (2010) | View |
| GLuc | Gaussia luciferase secretory signal peptide | Allows the tagged protein to be secreted from cytosol. | Microb Cell Fact. 14:20 (2015) | View |
| IL2-sig | Signal peptide of Homo sapiens interleukin 2 | Facilitates secretion of proteins. | Protein Expr Purif.118:105-12(2016) | View |
| HA-sig | The cleavable signal sequence of influenza hemagglutinin | Enhances localization of proteins to the endoplasmic reticulum membrane or plasma membrane. | J Biol Chem. 267:21995 (1992); Biochem Biophys Res Commun. 409:544 (2011) | View |
检测标签
| 姓名 | 描述 | Application Notes | References | 序列 |
|---|---|---|---|---|
| TC | Tetracysteine tag | Facilitates detection of tagged protein by biarsenical compounds such as FlAsH-EDT2 and ReAsH-EDT2, which turn fluorescent upon binding to the tag. | Nat Protoc. 5: 1666 (2010) | View |
| ProS2 | Protein S of Myxococcus xanthus | Facilitates detection of tagged protein by anti-ProS2 antibody; increases the solubility and stability of tagged protein in bacteria. | J Mol Biol. 286:1533 (1999) | View |
| TF | E.coli trigger factor, a ribosome-associated chaperone | Assists protein folding; facilitates detection of tagged protein by anti-trigger factor antibody; increases the solubility of tagged protein. | Biochim Biophys Acta. 1803:650 (2010) | View |
| Lifeact | A 17-aa F-actin-binding sequence derived from Saccharomyces cerevisiae | Allows visualization of F-actin dynamics in cells; can be used as a universal marker for F-actin imaging or as an alternative to phalloidin. | Nat Methods. 5:605 (2008) | View |
| SNAP | A 20 kDa engineered variant of the human DNA repair protein O-6-alkylguanine-DNA alkyltransferase, optimized for mammalian expression | Allows to observe localization and translocation of SNAP-tag fusion proteins; reacts specifically and rapidly with benzylguanine (BG) and benzylchloropyrimidine (CP) derivatives, leading to irreversible covalent labeling of the SNAP-tag with a wide variety of synthetic probes in live cells. CLIP and SNAP fusion proteins can be simultaneously and specifically labeled with different synthetic probes within cells. | Nat Biotechnol. 21:86 (2003); Methods Mol Biol. 1266:107 (2015) | View |
| CLIP | An engineered variant of the human DNA repair protein O-6-alkylguanine-DNA alkyltransferase, optimized for mammalian expression | Allows to observe localization and translocation of CLIP-tag fusion proteins; reacts specifically and rapidly with O2-benzylcytosine (BC) derivatives, leading to irreversible covalent labeling of the CLIP-tag with a wide variety of synthetic probes in live cells. CLIP and SNAP fusion proteins can be simultaneously and specifically labeled with different synthetic probes within cells. | Chem Biol. 15:128 (2008) | View |
| ACP | Acyl carrier protein of E. coli | Allows to observe localization and translocation of ACP-tag fusion cell surface proteins which are specifically labeled with CoA derivatives through post-translational modifications catalyzed by the phosphopantetheinyl transferase AcpS. | J Am Chem Soc. 126:8896 (2004) | View |
| HiBiT | A small 11 amino acid peptide that binds LgBiT with high affinity | Facilitates detection and monitoring of tagged endogenous protein in live cells; capable of producing highly active Nanoluc luciferase through high affinity complementation with LgBiT. | Biochem Biophys Rep. 12: 40 (2017); Chem Biol. 13:467 (2018) | View |
降解决定子标签
| 姓名 | 描述 | Application Notes | References | 序列 |
|---|---|---|---|---|
| DD | Destabilizing domain, mutant derived from human FKBP12 protein. | Targets the tagged protein for proteosomal degradation; addition of a compound, Shield-1, can stabilize the protein and prevents degradation. | Bioorg Med Chem Lett. 18:5941 (2008); PLoS One. 4: e6529 (2009) | View |
| S4D | Sal-like protein 4 (SALL4) degron (S4D) tag | Allows degradation of S4D-tagged proteins in the presence of immunomodulatory drugs (IMiDs) which interact with cereblon (CRBN) in the cullin E3 ubiquitin ligase complex to induce degradation of neosubstrates. | Commun Biol. 3:515 (2020) | View |
二聚体结构域
| 姓名 | 描述 | Application Notes | References | 序列 |
|---|---|---|---|---|
| FKBP(F36V) | FK506-binding protein 12 with F36V mutation | Promotes dimerization of tagged proteins in the presence of dimerizer ligands, e.g. AP20187. | Chem Rev. 110:3315 (2010); Sci Rep. 4: 6127 (2014) | View |
细胞穿透肽
| 姓名 | 描述 | Application Notes | References | 序列 |
|---|---|---|---|---|
| TAT | Amino acids 48–60 of transactivator of transcription protein (Tat) from human immunodeficiency virus type 1 (HIV-1). | Directs tagged protein to pass through the plasma membrane of mammalian cells. | J Biol Chem. 276:5836 (2001); ACS Nano. 8:1972 (2014); Biomol Concepts. 8:131 (2017) | View |
| R8 | Octaarginine tag (8 tandem argenines). | Directs tagged protein to pass through the plasma membrane of mammalian cells. | J Biol Chem. 276:5836 (2001); ACS Nano. 8:1972 (2014); Biomol Concepts. 8:131 (2017) | View |
转录调控蛋白
| 姓名 | 描述 | Application Notes | References | 序列 |
|---|---|---|---|---|
| VP16 | VP16 transcriptional activator domain encoded by HSV | Activates gene expression, commonly used with other DNA binding proteins. | Cell. 149:1447 (2012) | View |
| VP64 | Tetrameric repeat of the minimal activation domain of VP16 | Strongly activates gene expression, commonly used with other DNA binding proteins. | Proc Natl Acad Sci U S A. 95: 14628 (1998); Proc Natl Acad Sci U S A. 97: 1495 (2000) | View |
| KRAB | Krüppel-associated box domain from human gene ZNF10 | Inhibits gene expression, commonly used with other DNA binding proteins. | Proc Natl Acad Sci U S A. 91:4509 (1994); Proc Natl Acad Sci U S A. 97: 1495 (2000) | View |
嵌合抗原受体结构域
| 姓名 | 描述 | Application Notes | References | 序列 |
|---|---|---|---|---|
| CD8-leader | Leader signal peptide of T-cell surface glycoprotein CD8 alpha chain | Directs transport and localization of the protein to the T-cell surface. | Cell. 64:891 (1991); WO2014055771A1; US20160185862A1; US10144770B2 | View |
| CD8-hinge | Hinge region of T-cell surface glycoprotein CD8 alpha chain | Connects scFv and the transmembrane region; enhances tumor recognition efficiency; improves the expansion of CAR-T cells. | Cell. 64:891 (1991); J Immunother. 28:203 (2005); BMJ Open. 6: e013904 (2016); J Hematol Oncol. 10: 68 (2017); Oncoimmunology. 7: e1368604 (2018) | View |
| CD28-hinge | Hinge region of the T-cell surface glycoprotein CD28 | Connects scFv with the transmembrane region providing stability and flexibilty for efficient CAR expression and function; enhances efficiency of tumor recognition; improves expansion of CAR-T cells. | J Immunother. 32:689 (2009); Mol Ther. 25:2452 (2017) | View |
| CD8-TM | Transmembrane region of T-cell surface glycoprotein CD8 alpha chain | Anchors the CAR to the plasma membrane, bridges the extracellular hinge and antigen recognition domains with the intracellular signaling region. | Cell. 64:891 (1991); BMJ Open. 6: e013904 (2016); Oncoimmunology. 7: e1368604 (2018); WO2014055771A1; US20100105136A1 | View |
| CD28-TM | Transmembrane region of T-cell-surface glycoprotein CD28 | Anchors the CAR to the plasma membrane; enhances receptor expression and stability. | Hum Gene Ther. 24:259 (2013); Cancer Res. 74:6383 (2014); WO2014055771A1; US10144770B2 | View |