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Bacterial
Expression System >
Expresso ®
SUMO
產品特色
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Ready-to-use vectors
and competent cells - NO ligation step.
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High efficiency:
›90% recombinants.
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Tightly-controlled
expression of 6xHis-tagged-SUMO proteins
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Save time! Avoid
days of vector and cell preparation time.
Soluble
protein has never been this fast and easy!
With cleavable SUMO solubility tag
說明書下載
Datasheet
產品包裝
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5 Reactions |
10 Reactions |
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ExpressoTM
T7 SUMO Cloning and Expression System |
49003-1 |
49003-2 |
Kit
main contains: vector DNA, primer set, SUMO Express Protease, 10G
competent cells and BL21(DE3) competent cell
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Description |
Size |
Cat. No/ |
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HI-ControlTM
10G Chemically Competent Cells (SOLOs) |
12 transformations |
60110-1 |
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HI-ControlTM
1BL21(DE3) Chemically Competent Cells (SOLOs) |
12 transformations |
60435-1 |
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SUMO Express Protease |
200 units |
30801-2 |
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SUMO Cleavage Control Protein |
50 ug |
30805-1 |
pETiteTM N-His SUMO
Kan Vector Information
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Small size, 2.5 kb (VS. pET 5.4 kb)
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Based on Lucigen’s patented pSMART®
vectors.
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T7-lac promoter, RBS, start / stop
codon, SUMO
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Do not contain the lacZ alpha gene NO
blue / white colony screening
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Insert size: ≤12 kb
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Pre-linearized
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Wha's SUMO
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SUMO=Small Ubiquitin-like
MOdifier
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Derived from the yeast
SMT3 gene
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Can enhance the expression
and solubility of proteins
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6XHis-SUMO tag can be
cleavage by SUMO Express protease (at C-terminal)
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6XHis SUMO MW=12 kDa,
migrate ~15-18kDa
What's SUMO Express
Protease
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Derived from
the yeast ULP1 gene
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Highly
specific for the teritary structure of SUMO
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Cleavage of
6XHis-SUMO at C-terminal
Five-Second
Directional Cloning of PCR-Amplified Genes
Insertion of a gene
into the pETite N-His SUMO vector for expression
Forward primer: vector sequence includes
the last
18 nts of SUMO +21 nts of target gene
Reverse primer: vector sequence includes
Stop anticodon +21 nts of target gene
Rescue Insoluble
Protein with SUMO Protein Tag
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(A)
PCR products from 48 putative hydrolase genes ranging from
~1 to >3 kb. |
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(B)
Uninduced (-) and IPTG-induced (+) samples of HI-Control
BL21(DE3) cells with 6 different genes cloned into the
pETite C-His Vector. |
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(C)
Enhanced solubility of SUMO-tagged 2201 and 2442 gene
products. Total cell extract and soluble fractions are
shown. |
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(D)
Removal of 6xHis-SUMO tag from purified SUMO-2201 fusion
protein by SUMO protease. –prot: uncleaved SUMO-2201 fusion
protein after IMAC purification; +prot: SUMO
protease-treated fusion protein; C: isolated 2201 protein
after removal of 6xHis-SUMO fragment and SUMO protease by
subtractive IMAC. |
ExpressoTM
T7 SUMO Cloning and Expression System --- Data
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Fibrobacter succinogenes
gene number |
Soluble protein yield
w/o SUMO tag |
Soluble protein
w/SUMO tag |
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1425 |
0 mg/liter |
0 mg/liter |
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1765 |
0 mg/liter |
10 mg/liter |
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1793 |
0 mg/liter |
17 mg/liter |
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1994 |
0 mg/liter |
17 mg/liter |
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2201 |
0 mg/liter |
20 mg/liter |
Improvement of soluble protein yield
with SUMO tag. Yield of soluble protein was improved significantly
for 4 of 5 Fibrobacter succinogenes genes when cloned into pETite
N-His SUMO and expressed in HI-Control BL21(DE3) Cells. Cultures
were induced with 1mM IPTG and grown overnight at 22°C. Yields were
calculated from the amount of pure protein obtained from 100 ml of
cell culture after purification over a Ni-NTA column.
Cloning and expression Troubleshooting Guide
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PCR product
should be 10 ng/ul
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If template is
Kan-plasmid, suggest run and cut gel to purify PCR product
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Insert should
add 25-100 ng (1-3 ul)
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When transform,
all tube should pre-chilled
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Removal of SUMO
tag: Please do Dialysis then use SUMO protease cleavage
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