pDsRed-Express2-N1載體描述 pDsRed-Express2-N1 is a mammalian expression vector designed to express a protein of interest fused to the N-terminus of DsRed-Express2, a variant of the Discosoma sp. red fluorescent protein, DsRed (1). DsRed-Express2 retains the fast maturation and high photostability characteristic of its predecessor, DsRed-Express (2), and has been engineered (through additional amino acid substitutions) for increased solubility (3). Although it most likely forms the same tetrameric structure as wild-type DsRed, DsRed-Express2 displays a greatly reduced tendency to aggregate, resulting in reduced cyto- and phototoxicity, and making DsRed-Express2 much better suited for in vivo applications involving sensitive cells, such as primary or stem cells. (Please note: Because DsRed-Express2 likely forms tetramers, its suitability as a fusion partner will largely depend on how its tetramerization affects the function of the protein to which it is fused.) DsRed-Express2 also exhibits extremely low residual green fluorescence, which allows cells expressing the protein to be effectively separated from other fluorescently labeled cell populations by flow cytometry. The multiple cloning site (MCS) in pDsRed-Express2-N1 is positioned upstream of the DsRed-Express2 coding sequence. A Kozak consensus sequence (4), located between the MCS and the DsRed-Express2 coding sequence, enhances the translational efficiency of the unfused DsRed-Express2 protein in eukaryotic cells. SV40 polyadenylation signals downstream of the DsRed-Express2 coding sequence direct proper processing of the 3' ends of the DsRed-Express2 and fusion gene mRNA transcripts.
The vector backbone also contains an SV40 origin for replication in mammalian cells expressing the SV40 large T antigen, a pUC origin of replication for propagation in E. coli, and an f1 origin for single-stranded DNA production. This vector also has a neomycin-resistance cassette (Neor) that allows G418 selection of stably transfected eukaryotic cells (5). This cassette consists of the SV40 early promoter, a Tn5 kanamycin/neomycin resistance gene, and herpes simplex virus thymidine kinase (HSV TK) polyadenylation signals . A bacterial promoter upstream of this cassette allows kanamycin resistance in E. coli.
To construct a fusion protein, the gene of interest must be cloned into pDsRed-Express2-N1 so that it is in-frame with the DsRed-Express2 coding sequence; the gene must include an initiation codon (ATG), and lack in-frame stop codons. pDsRed-Express2-N1 can also be used as a cotransfection marker, as the unmodified vector will express DsRed-Express2 in mammalian cells.
pDsRed-Express2-N1 can be transfected into mammalian cells using any standard transfection method. Fusions that retain the fluorescence properties of the native DsRed-Express2 protein (excitation and emission maxima: 541 and 591, respectively) can be monitored by flow cytometry and localized by fluorescence microscopy. If required, stable transfectants can be selected using G418.
For Western analysis, DsRed-Express2 can be detected with either the Living Colors DsRed Polyclonal Anti-body (Cat. No. 632496) or the Living Colors DsRed Monoclonal Antibody (Cat. Nos. 632392 and 632393). pDsRed-Express2-N1載體含有以下元件： PCMV IE (human cytomegalovirus immediate early promoter): 1–589
MCS (multiple cloning site): 591–671
DsRed-Express2 (Discosoma sp. red fluorescent protein variant)
Kozak consensus translation initiation site: 672–682
Start codon (ATG): 679–681; Stop codon: 1354-1356
SV40 early polyA+ signals: 1508–1513 & 1537–1542; mRNA 3' ends: 1546 & 1558
f1 origin of replication: 1605–2060 (complementary)
SV40 origin of replication: 2401–2539
Kanr/Neor (kanamycin/neomycin resistance gene)
Neomycin phosphotransferase coding sequences:
Start codon (ATG): 2585–2587; stop codon: 3377–3379
pUC origin of replication: 3964–4607 Propagation in E. coli Recommended host strain: DH5α, HB101, and other general purpose strains.
Selectable marker: plasmid confers resistance to kanamycin (50 μg/ml) in E. coli hosts.
E. coli replication origin: pUC
Copy number: high
Plasmid incompatibility group: pMB1/ColE1 Excitation and emission maxima of DsRed-Express2 Excitation maximum = 554 nm
Emission maximum = 591 nm
