原核蛋白表达
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Solving Problems in Bacterial Protein Expression
vector
host
growth conditions
Page 8
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pETcoco™
Page 12
Tuner™(DE3) cells carrying pETcoco TM-1 containing
Ann. Rev. Genet
Page 18
Page 21
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pETcoco™
inNovations 15)
Page 24
Page 25
Page 27Page 28
124
3
Page 32
host vector
growth conditions
Page 44
Cells were grown at 37C to OD 600of 0.8 and induced with 1mM IPTG for 2.5 h. Total cell protein samples were electrophoresed in a 4-20%acrylamide gel.
inNovations Cells were grown at 37C to OD 600of 0.8 and induced with 1mM IPTG for 2.5 h. Total cell protein samples were electrophoresed in a 4-20% acrylamide gel.
Page 46
J . Biol . Chem . 272: 15661 (1997)
Wild type trx B
-
trx B -gor
-
Effect of consecutive CGG rare codons on target protein
expression
A pET-15b recombinant plasmid containing five consecutive
CGG codons near the 5’end of the β-gal coding region was
transformed into Rosetta™(DE3) amd Rosetta 2(DE3) cells.
The cells were induced with IPTG for 3 hr and harvested by
centrifugation.
Page 49Page 50
Vector Host
Growth Conditions
Page 60SDS-PAGE analysis of eukaryotic target proteins purified from cultures induced with Overnight Express verses IPTG
SDS-PAGE analysis of crude and purified proteins from cultures induced with Overnight Express verses IPTG
50 ml portions of induced BL21(DE3) containing
pET-41a(+) (GST) were harvested by centrifugation and
resuspended in 2 ml PBS (1), another lysis reagent (2),
or BugBuster® Reagent (3). The sample in PBS was
lysed by sonication. Extracts were clarified by
Page 64
•T7•Tag sequence
Page 67
chromatography Magnetic h
Page 73
BL21(DE3) cells containing pET-41b(+)
(GST /His•Tag sequence) were induced
with 1mM IPTG for 3 hr, lysed
culture medium with PopCulture™ Reagent
and Benzonase®Nuclease and purified on
magnetic IDA resin (lane 2).
inNovations magnetic-based
magnetic stand and remove
Page 76
M 1 2 3 4 5 6 M
Biotinylated Thrombin digestion Page 81
host vector
growth conditions