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Bacterial
Expression System >
Expresso ®
Rhamnose
Expresso ® Rhamnose Cloning and
Expression System
產品特色
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pRhamTM vectors
with ttunable rhaPBAD promoter.
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6X His of N- /
C- / 6X His-SUMO.
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One competent
cell strain is provided for cloning and expression.
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Auto-induction
reagents included.
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For
High-throughput protein expression
說明書下載 Datasheet
產品包裝
|
Product |
Size |
Cat. No. |
|
Expresso® Rhamnose Cloning and Expression System, C-His |
5 rxns |
49012-1 |
|
|
10 rxns |
49012-2 |
|
Expresso® Rhamnose Cloning and Expression System, N-His |
|
|
|
|
|
|
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Expresso® Rhamnose Cloning and Expression System, N/C-His
Combo, 5rxns of each of N-/C-His |
10 rxns |
49010-1 |
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Expresso® Rhamnose SUMO Cloning and Expression System |
5 rxns |
49013-1 |
|
|
10 rxns |
49013-2 |
|
Glucose Solution, 15% w/v |
5 X 1.25 mL |
49022-1 |
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Rhamnose Solution, 20% w/v |
5 X 1.25 mL |
49021-1 |
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SUMO Cleavage Control Protein |
50 ug |
30805-1 |
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SUMO Express Protease |
200 U |
30801-2 |
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E. Cloni®
10G Chemically Competent Cells (SOLOs) |
12 rxns |
60106-1 |
rhaPBAD
promoter
•rhaPBAD
promoter replacing the T7 promoter.
•Because
rhaPBAD
is recognized by the bacterial RNA polymerase, so just
single-host is used for both clone construction and protein
expression.
•Absence
L-rhamnoseà
allow stable clone.
construction.
à
toxic gene products.
•RhaR
and RhaS are activatorsà
bind rhamnose.
•rhaPBAD
tunable
à
increase difficult or toxic protein expression levels.
rhaPBAD Vectors
|
Low copy number (~20 copies/cell)
Yield: 0.5-1.0 ug plasmid /mL |
 |
Induction of
Protein Expression
•Recommended:
–Small
scale expression trials, 2-50mL
–Maximal
induction: 0.2% rhamnose, 37 degree 8hr (or 24 hours at 20
to 30 degree)
–For
improve solubility of protein: 0.001% to 0.1% rhamnose.
Tuning recombinant protein expression levels with rhamnose
induction.
|
The pRham C-His Kan Vector
containing a gene encoding a blue fluorescent protein (BFP)
was transformed into E. cloni 10G cells. An uninduced
starter culture was inoculated to a starting OD600 of 0.8
into culture tubes containing LB media with 30 µg/ml
kanamycin and the indicated concentrations of rhamnose (0 to
0.2% w/v) or 2% glucose. After overnight incubation at 37°C,
samples were harvested by centrifugation, lysed in SDS-PAGE
loading buffer, and analyzed by SDS-PAGE. The Coomassie-blue
stained gel shows total cellular protein. Protein expression
levels are responsive to rhamnose concentrations between
0.001% and 0.2%. |
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Cloning and expression Troubleshooting Guide
•Very
few or no transformants
–Toxic
gene product
•à
use plates containing 0.5% glucose to prevent leaky expression.
•à
Incubate plates at room temperature.
•Low
recovery of recombinant protein
–Recombinant
protein expressed in inclusion bodies
•Incubate
culture at a lower temp. (20 to 30degree)
•Lower
conc. Rhamnose
•Use
SUMO system
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