pGEMEX®-1 and pGEMEX-2 Vectors BioVector NTCC质粒载体菌种细胞基因保藏中心

pGEMEX®-1 and pGEMEX-2 Vectors BioVector NTCC质粒载体菌种细胞基因保藏中心
The pGEMEX® Vectors(a,b) are T7 expression vectors that can be used for cloning,
as templates for in vitro transcription and for production of single-stranded DNA
(ssDNA). Expression from the pGEMEX® Vectors is based on the T7 expression system(
a) developed by Studier (1), which uses a convenient vector/host combination for
high-level expression of cloned genes in vivo. Sequences cloned into the pGEMEX®
Vectors are expressed as T7 gene 10 fusion proteins in JM109(DE3)(a) or
BL21(DE3)pLysS(c) host strains containing an inducible gene for T7 RNA polymerase.
JM109(DE3) is a specially constructed host strain that contains an IPTGinducible
gene for T7 RNA polymerase; JM109(DE3) and JM109 are supplied with
the pGEMEX® Vectors. The pGEMEX®-1 and pGEMEX®-2 Vectors differ by only two
base pairs resulting in a shift in the reading frame (Figure 1). The bacteriophage T7
gene 10 leader peptide (260 amino acids) is expressed very efficiently in this host
and can accumulate to greater than 50% of total cell protein in three hours or less.
Typical levels of fusion protein production are on the order of 10mg per liter of
induced culture.
The pGEMEX® Vectors are unique in that they contain three RNA polymerase promoters.
The T7 promoter, along with the gene 10 ribosome binding site, is positioned
upstream from the gene 10 leader fragment. A T7 transcription terminator is incorporated
downstream from the multiple cloning region and functions to end transcription flank the multiple cloning region, allowing the production in vitro of high specific
activity single-stranded RNA probes and substrate amounts of RNA from either
strand of a cloned insert (Figure 2).
In-frame subcloning from lambda expression vectors into the pGEMEX® Vectors is
easily performed. The reading frame of the pGEMEX®-1 Vector at the EcoR I cloning
site is identical to that of λgt11. These plasmids also contain the f1 origin of replication
and thus can be used to generate ssDNA for sequencing or in vitro mutagenesis.
Complete protocols for cloning and generating RNA using pGEMEX® Vectors
can be found in the Riboprobe® In Vitro Transcription Systems Technical
Manual(c,d) #TM016. Protocols for cloning and generating ssDNA using pGEMEX®
Vectors can be found in reference 2.
For small-scale M13 template preparation, Promega’s Streptavidin MagneSphere®
Paramagnetic Particles plus M13 Oligo (Cat.# Z5392) is a more rapid and simple to
perform method than traditional procedures for M13 template production, such as
PEG/phenol procedures, which produce microgram quantities of DNA from milliliter volumes
(4). These traditional procedures are difficult to scale down and require many
centrifugation steps for the separation of phenol layers and the precipitation of DNA.
While smaller scale M13 DNA purification methods have been developed, these either
use specialized filtration steps or PEG/SDS extractions with multiple centrifugation
steps
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