pDual-GC载体介绍 The pDual-GC vector, which is based on Agilent’s pDual expression vector, is designed for high-level protein expression in mammalian and bacterial cells (see Figure 1). The vector contains the promoter and enhancer region of the human cytomegalovirus (CMV) immediate early gene‡ for constitutive expression of the clones in either transiently or stably transfected mammalian cells. Inducible gene expression in prokaryotes is directed from the hybrid T7/lacO promoter; the vector carries a copy of the lac repressor gene (lacIq), which mediates tight repression in the absence of isopropyl-β-D-thio-galactopyranoside (IPTG). Expression is therefore regulated using IPTG in bacteria that contain the T7 RNA polymerase, for example, BL21(DE3) bacterial cells. A tandem arrangement of the bacterial Shine-Dalgarno1 and mammalian Kozak2 ribosomal binding sites (RBS)allows for efficient expression of the ORF in both bacterial and mammalian systems.
The unique cloning region of the pDual GC expression vector is characterized by the presence of two Eam1104 I recognition sequences (CTCTTC) directed in opposite orientations and separated by a spacer region encoding the β-lactamase gene with a prokaryotic promoter.Digesting the vector with the Eam1104 I restriction enzyme creates a 3-nucleotide 5´ overhang that is complementary to the translation initiation codon (ATG) of the DNA insert.
Inserts must be generated by PCR amplification with primers that contain Eam1104 I recognition sites and a minimal flanking sequence at their 5´ termini. The ability of Eam1104 I to cleave several bases downstream of its recognition site allows the removal of superfluous, terminal sequences from the amplified DNA insert. The elimination of extraneous nucleotides and the generation of unique, nonpalindromic sticky ends permit the formation of directional seamless junctions during the subsequent ligation to the pDual GC expression vector.
3 In both bacterial and mammalian cells, the dominant selectable marker is the neomycin phosphotransferase gene, which is under the control of the β-lactamase promoter in bacterial cells and the SV40 promoter in mammalian cells. Expression of the neomycin phosphotransferase gene in mammalian cells allows stable clone selection with G418, whereas in bacteria the gene confers resistance to kanamycin selection. All pDual GC clones express a fusion protein consisting of the cDNA, a thrombin cleavage site, three copies of the c-myc epitope tag, and a single copy of the 6×His purification tag. The c-myc epitope is derived from the human c-myc gene and contains 10 amino acid residues (EQKLISEEDL).
4 This allows for convenient and sensitive detection of expressed proteins with anti–c-myc antibody. The 6×His purification tag consists of six histidine residues and allows for quick and easy purification of the fusion protein from bacterial cells.
5 A thrombin cleavage site between the protein encoded by the cDNA and the c-myc and 6×His tags allows the removal of both tags when desired, for example, following protein purification.
pDual Expression Vectors - Details & Specifications
The pDual expression vector directs expression of heterologous genes in both mammalian and prokaryotic systems. For constitutive expression in mammalian cells, the pDual expression vector contains a mutagenized version of the promoter/enhancer of the human cytomegalovirus (CMV) immediate early gene. Inducible gene expression in prokaryotes is directed from the hybrid T7/lacO promoter; the pDual expression vector carries a copy of the lac repressor gene (lacIq), which mediates tight repression in the absence of isopropyl- ß -D-thio-galactopyranoside (IPTG).
Efficient translation of mRNA generated in either the mammalian or prokaryotic system is achieved by a tandemly arranged Shine-Dalgarno/Kozak consensus sequence. In both bacterial and mammalian cells, the dominant selectable marker is the neomycin phosphotransferase gene which is under the control of the ß-lactamase promoter in bacterial cells and the SV40 promoter inmammalian cells (Figure 1). Expression of the neomycin phosphotransferase gene in mammaian cells allows stable clone selection with G418, whereas in bacteria the gene confers resistance to kanamycin selection.
Figure 1. Western Blot Analysis of Transfected Mammalian Lysates From CHO cell lysates, 1 mg of protein was electrophoresed on a 4-20% tris-glycine-SDS gel, then transferred to a nitrocellulose membrane, and reacted with antiluciferase (A) or anti-c-myc (B) antibodies. Antibody binding was detected using chemiluminescence methods. The difference in molecular weights of the different detected proteins is due to the use of epitope and purification tags. Estimated molecular weights: pDual GC + Luciferase is 68 kDa, pCMV-Tag5 + Luciferase is 63 kDa, and luciferase protein is 61 kDa. The difference in chemiluminescent signal between pDual GC + Luciferase and pCMV-Tag5 + Luciferase (B) is probably due to the presence of three copies of the c-myc epitope in the pDUAL GC vector and only one copy of the c-myc epitope in the pCMV-Tag5 vector. The lower molecular weight band that is detected at about 40 kDa with the anti-c-myc antibody (B) was also detected in control cells that had notThe unique cloning region of the pDual expression vector is characterized by the presence of two Eam1104 I recognition sequences (CTCTTC) directed in opposite orientations and separated by a spacer region encoding two EcoR I sites. Digesting the vector with the Eam1104 I restriction enzyme creates a 3-nucleotide 5´ overhang that is complementary to the translation initiation codon (ATG) of the DNA insert.
Inserts must be generated by PCR amplification with primers that contain Eam1104 I recognition sites and a minimal flanking sequence at their 5´ termini. The ability of Eam1104 I to cleave several bases downstream of its recognition site allows the removal of superfluous, terminal sequences from the amplified DNA insert. The elimination of extraneous nucleotides and the generation of unique, nonpalindromic sticky ends permit the formation of directional seamless junctions during the subsequent ligation to the pDual expression vector.
The pDual vector contains the Calmodulin Binding Peptide (CBP) affinity tag, located 3´ to the cloning site, for optional fusion of the affinity tag to the carboxy terminus of the protein-coding sequence of interest. The CBP-affinity tag is preceded by a thrombin cleavage site which allows the removal of the fusion tag from the protein of interest.
