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ImpactVector™
The Technology
The need for high level, tissue-specific,
regulated expression of a gene arises
in all types of plant genetic research
and can be crucial in obtaining the
unequivocal results you need for
shorter time-to-publish or
time-to-market.
To help you realize these goals,
ImpactVector™ combines the
strongest green tissue promoter (over
7% of total soluble protein) with the
most versatile targeting options
available to date (5 different
subcellular localizations). Each vector
also comes optionally equipped with a
c-myc antibody tag for easy
identification of your protein and with a
His-tag for purification.
The universal multiple cloning site
allows one-step cloning for all different
targeting options. The 8-cutter
AscI-PacI restriction sites allow easy
subcloning to pBINPLUS or any other
plant expression vector of choice.
The RbcS1 promoter
The strength of the promoter
Ribulose bisphosphate carboxylase (RBC) is the primary enzyme of the
carbon fixation process. It constitutes approximately 50-60% of total leaf
protein, and is the most abundant protein on earth. RBC is composed of 8
small subunits of 14 kDa (rbcS) encoded by a gene family of 2-12 genes on the
nuclear genome and 8 large subunits of 55 kDa (rbcL) encoded by a single
gene on the chloroplast genome. RbcS genes contribute 10-12% of total
soluble protein, so that similar expression levels would be expected from
constructs using such an RbcS promoter. Yet in the past 15 years RbcS
expression vectors never exceeded 1% of total soluble protein.
A gene expression cassette ImpactVector1 based on a rubisco small subunit
promoter from the Asteraceous chrysanthemum has been designed which is 8
times stronger than the commonly used dCaMV-35SEnh promoter. The
expression cassette preserved 1 kb of the native RbcS1 terminator sequence,
which may contribute to the improved performance relative to similar RbcS
promoters derived from other plants (Outchkourov et al. 2003)
Light regulated expression
The expression of genes cloned into the ImpactVector1 expression cassette is
light-dependent. This property is an advantage as it allows a time dependent
study of the fate of both transcript and protein (Outchkourov et al. 2003).
Uniform high level expression
Figure 1 shows that 90% (19/21) of all tobacco-GUS transgenics express
within a range of less than one order of magnitude (1-10% TSP). This
suggests that position effects on gene expression are negligible when this
construct is used.
Tested in different plants and with different genes
The ImpactVector1 (RbcS1)
expression cassette was
shown to yield average
GUS expression levels of
up to 800 nmol/mg.min in
tobacco. In chrysanthemum,
from which the promoter
was isolated, expression
levels were lower, but still
superior to any other
reported
promoter.
Equistatin was expressed at
7% in potato, cystatin at 3%
in tomato and a Bt toxin
hybrid, which is notorious for low expression levels, at 0.3% in potato and the monocot
garlic. These results illustrate that expression levels are always strongly gene and plant
species dependent. To obtain expression levels in the range of 10% or more it may be
necessary to optimize the genes first for codon usage and other instability factors. Still the
resulting protein may be the target of degrading plant proteases. Changing the targeting
of the protein may then result in strong improvements of the quality and level of protein
expression (Outchkourov et al., 2003)
PRI ImpactVector™ 台灣區獨家代理
艾特克生物科技股份有限公司
TEL:02-29072616
FAX:02-29072816