pDsRed-Express2載體描述 pDsRed-Express2 is a prokaryotic expression vector that encodes 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 and reduced cytotoxicity (3). Although it most likely forms the same tetrameric structure as wild-type DsRed, DsRed-Express2 displays a greatly reduced tendency to aggregate; this results in minimal cytotoxicity, which makes DsRed-Express2 much better suited for in vivo applications involving sensitive cells, such as primary or stem cells. 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.
In pDsRed-Express2, the DsRed-Express2 coding sequence is flanked by separate and distinct multiple cloning sites (i.e., the 5' MCS and 3' MCS) that make it easy to excise the gene for use in other cloning applications. In E. coli, DsRed-Express2 is expressed from the lac promoter (Plac) as a fusion with several amino acids, including the first five amino acids of the LacZ protein. Note, however, that if the DsRed-Express2 coding sequence is excised using a restriction site in the 5' MCS, the protein will no longer be expressed as a fusion (as it is when it is expressed from the lac promoter). A Kozak consensus sequence is located immediately upstream of the DsRed-Express2 coding sequence to enhance translational efficiency in eukaryotic cells (4).The entire DsRed-Express2 expression cassette in pDsRed-Express2 is supported by a pUC19 backbone, which contains a high-copy number origin of replication and an ampicillin resistance gene (Ampr) for propagation and selection in E. coli.
pDsRed-Express2 is primarily intended to serve as a source of DsRed-Express2 cDNA. The flanking MCS regions make it possible to excise the DsRed-Express2 coding sequence and insert it into other vector systems. The vector can also be used to express DsRed-Express2 in bacteria. Cells expressing DsRed-Express2 (excitation and emission maxima: 554 nm and 591 nm, respectively) can be detected by either fluorescence microscopy or flow cytometry 8–12 hours after transfection.
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載體含有以下元件： Plac (lac Promoter): 95–178
CAP binding site: 111–124
–35 region: 143–148; –10 region: 167–172
lac operator: 179–199
lacZ-DsRed-Express2 fusion expressed in E. coli
Ribosome binding site: 206–209
Start codon (ATG): 217–219; Stop codon 961–963
5' MCS (5' multiple cloning site): 234–292
DsRed-Express2 (Discosoma sp. red fluorescent protein variant)
Kozak consensus translation initiation site: 282–292
Start codon (ATG): 289–291; Stop codon: 964–966
3' MCS (3' multiple cloning site): 966–1065
Ampr (Ampicillin resistance gene): 1511–2371
pUC origin of replication: 2519–3161 Propagation in E. coli Recommended host strain: DH5α
Selectable marker: plasmid confers resistance to ampicillin (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
