商品编号


60810-2



品牌


Lucigen



公司


Lucigen



公司分类


Low Endotoxin Cells




24 rxns (DUOs)







商品信息

Eliminate endotoxin at the source


Genetically modified LPS does not trigger endotoxic response in human cells

Ideal for mammalian cell immunogenicity testing, toxicity assays, and therapeutic protein drug discovery

Useful for membrane and lipid binding protein production

Protein expression similar to BL21(DE3) cells without requiring downstream endotoxin removal

Reduce false positives in cytokine assays, improve confidence in your results

Frequently Asked Questions







Introduction

Genotype information

Why endotoxin removal is not the best method

Protein expression with ClearColi

Endotoxicity assay results

LAL testing: does it really measure endotoxin?

Growth rates for ClearColi BL21(DE3) cells

Useful reference articles

ClearColi licensing information



Introduction to ClearColi? technology:

Is there a better way to eliminate endotoxin contamination?
Now there is.?

Instead of removing lipopolysaccharide (LPS) contamination from your protein or plasmid DNA preparations, eliminate the LPS at the source. ??Genetically modified LPS from a novel
E. coli
strain produces functionally clean recombinant proteins and plasmids.? ClearColi? BL21(DE3) cells are the first commercially available competent cells with a modified LPS (Lipid IV
A
- see Fig. 1) that does not trigger the endotoxic response in mammalian cells. ClearColi? cells lack outer membrane ago
NIST
s for hTLR4/MD-2 activation; therefore, activation of hTLR4/MD-2 signalling by ClearColi is several orders of magnitude lower compared with
E. coli
wild-type cells, and heterologous proteins prepared from ClearColi are virtually free of endotoxic activity. After minimal purification from ClearColi cells, proteins or plasmids, which may still contain Lipid IV
A
, can still be used in most applications without eliciting an endotoxic response (see Endotoxicity assay results for details).









Figure 1. The LPS of a normal
E. coli
cell compared to the genetically modified Lipid IV
A
from ClearColi cells.
? In ClearColi, the oligosaccharide chain has been deleted, and two of the six acyl chains have been removed to disable the endotoxin signal.




Modifications to the genotype of the ClearColi cells consist of seven separate gene deletions, thereby ensuring there is no chance of genetic reversion back to wild type and production of normal LPS.? These mutations result in the deletion of the oligosaccharide chain from the LPS, making it easier to remove the resulting lipid IV
A
from the downstream product.? More importantly, two of the six acyl chains are deleted.? The six acyl chains of the LPS are the trigger which is recognized by the Toll-like receptor 4 (TLR4) in complex with myeloid differentiation factor 2 (MD-2), causing activation of NF-?B and production of proinflammatory cytokines.? Lipid IV
A
, which contains only four acyl chains, is not recognized by TLR4 and thus does not trigger the endotoxic response (see Fig. 2).









Fig. 2. Comparison of relative NF-κB induction in HEK-Blue Cells using purified LPS from a K-12
E. coli
strain or from pure, synthetically manufactured Lipid IV
A
.




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Genotype Information:

ClearColi BL21(DE3) competent cells have the following genotype:
F–
ompT hsdSB (rB- mB-) gal dcm lon
&lam
BD
a;(DE3 [
lacI

lac
UV5-T7 gene 1
ind1 sam7 nin5
])
msbA148
Δ
gutQ
Δ
kdsD
Δ
lpxL
Δ
lpxM
Δ
pagP
Δ
lpxP
Δ
eptA

Seven specific deletion mutations (Δ
gutQ
Δ
kdsD
Δ
lpxL
Δ
lpxM
Δ
pagP
Δ
lpxP
Δ
eptA
) encode the modification of LPS to Lipid IV
A
, while one additional compensating mutation (
msbA148
) enables the cells to maintain vi
ABI
lity in the presence of the LPS precursor lipid IV
A
.

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Why Endotoxin Removal is not the Best Method

Lipopolysaccharide (LPS), also known as endotoxin, is a potent ago
NIST
for hTLR4/MD-2-mediated proinflammatory activity in human immune cells. To prevent toxicity, recombinant proteins, commonly manufactured in
E. coli
, must be essentially free of endotoxin. However, efficient elimination of endotoxin is a challenging task, and none of the downstream applications remove endotoxin entirely. Current methods for endotoxin removal from recombinant proteins are varied, including ultra-filtration, activated carbon, surfactants, anion exchange chromatography, and immobilized sepharose.? Each of these strategies involves negative effects: significant yield loss, high cost, loss of bioactivity of the protein, or bioactivity of the additives used for endotoxin cleanup.? Table 1 below describes some of the most common methods and their associated shortcomings:




Table 1:

?





?Method




Disadvantages






Ultrafiltration




Only useful for small proteins

Endotoxin monomers may permeate the membrane due to endotoxin/protein interaction

Inefficient for proteins which can be damaged by physical forces







Activated carbon




Adsorbing activity for both endotoxin and protein







Surfactants




Expensive

May affect bioactivity of protein

Difficult to completely remove

Removal may lead to product loss







Anion-exchange chromatography




High adsorbtion of both endotoxin and acidic protein

No selectivity to adsorb endotoxin







Histamine- and histidine-immobilized Sepharose




Removing capacity dependent on the ionic strength


BIOLOG
ical activity of histamine







Polymyxin B-immobilized Sepharose




Protein losses due to the ionic interaction between polymyxin B and protein

Polymixin B is physiologically active






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Protein Expression with ClearColi

The relative production efficiency of endotoxin free ClearColi? BL21(DE3) competent cells was compared to normal BL21(DE3) cells using two different recombinant proteins.? Although overall growth rates of the ClearColi are slower, final protein production levels are very similar (see Fig. 3 and 4).









Figure 3. Comparison of protein expression in ClearColi BL21(DE3) and
Lucigen
's E. Cloni? EXPRESS BL21(DE3) competent cells.
Cells containing a T7 expression plasmid harboring a gene encoding the human apolipoprotein A1 (ApoA1) were grown in LB Miller medium at 37°C. When cultures reached OD
600
of 0.6 to 0.8, expression was induced by the addition of 0.4 mM IPTG and incubation was continued for 3 hours. Equivalent numbers of uninduced (-) and induced (+) cells were lysed by heating in Laemmli buffer and samples were analyzed by SDS-PAGE on a 4% - 20% polyacrylamide gr
ADI
ent gel.












Figure 4. Comparison of fluorescent protein expression in ClearColi BL21(DE3) and
E. cloni
EXPRESS BL21(DE3) cells.
?A pET expression vector harboring a gene encoding a yellow fluorescent protein (LucY) under the control of a T7 promoter was transformed into both ClearColi BL21(DE3) and E. cloni EXPRESS BL21(DE3). Colonies were inoculated into LB-Miller medium and grown at 37°C for induction. Samples of induced and uninduced cells containing equivalent numbers of cells were centrifuged, and cell pellets were photographed under long-wavelength UV
Illumina
tion.




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Endotoxicity Assay Results

In most cases, a simple nickel column purification is sufficient for endotoxic shock response negative recombinant proteins suitable or downstream eukaryotic cellular response? assays.? Researchers can now assay target protein effects without concern that immune response triggers are a result of LPS endotoxin contamination.? To demonstrate, ApoA1 protein expressed from ClearColi BL21(DE3) competent cells was Ni-column purified and tested for endotoxicity using both the HEK-Blue cell line from
InvivoGen
(see Fig 5) ?and the LAL assay (see Fig. 6).









Figure 5. Comparison of endotoxic response from protein derived from ClearColi BL21(DE3) and tr
ADI
tional BL21(DE3) competent cells?




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LAL Testing: Does it really measure endotoxin?


Limulus
amebocyte assay testing is an FDA-approved method for detection of endotoxins and the most common assay used; however the LAL assay is activated solely by the 4?-monophosphoryldiglucosamine backbone of LPS.? LAL activity is minimally influenced by acylation pattern of LPS, the key determinant of endotoxicity in eukaryotic cells.? The LAL assay also recognizes a wider spectrum of LPS/lipid A variants than the central cellular endotoxin sensor system of the human immune cell system.? As such, false positive results can and will result due to the lack of specificity of the assay.

A simple Ni-column purification step for proteins produced from ClearColi cells will reduce LAL response levels by 95% or greater (see Fig. 6).? However, the residual EU measurements are due to the non-specific nature of the assay unless extraneous LPS contamination from other sources is present.? Alternative toxicity assays, such as those using HEK-Blue cells (see Fig. 5) suggest that even in the presence of EU levels above threshholds normally targeted by researchers, the actual immunogenic effects from ClearColi-derived proteins are non-existent.

Due to the non-specificity of the LAL assay when combined with lipid IV
A
from ClearColi, it is suggested that researchers consider alternative methods of endotoxin measurement.









Figure 6. Comparison of ?endotoxin units as measured by the LAL assay using nickel-column purified recombinant ApoA1 and HSP protein expressed in ClearColi BL21(DE3) (red bars) and E. cloni EXPRESS BL21(DE3) (grey bars) competent cells.?
Protein expressed from ClearColi demonstrates significant reduction in EU/mg without endotoxin removal steps
.




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Growth Rates for ClearColi BL21(DE3) Cells

ClearColi BL21(DE3) cells grow at approximately 50% of the rate of normal BL21(DE3) cells (see Fig. 7).? Users should expect to see very small colonies for the first 24 hours after plating transformants.?
Lucigen
recommends incubating plates for 32-40 hours before picking colonies for future experiments.? When grown to sufficient densities, ClearColi BL21(DE3) cells produce similar protein levels as normal BL21(DE3) cells when comparing equal numbers of cells.









Figure 7. Comparison of growth rates for ClearColi BL21(DE3) vs.
E. cloni
EXPRESS BL21(DE3) cells.
Cells were inoculated to an initial OD
600
of ~0.003 in 200 ml of LB Miller medium and grown at 37° C with shaking at 210 rpm. The OD
600
?of the cultures was recorded hourly.




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Relevant Reference Articles:


Teghanemt, et al, Molecular Basis of Reduced Potency of Underacylated Endotoxins,
J Immunol
, 2005;
175:
4669-4676

Mamat, et al, Single amino acid substitutions in either YhjD or MsbA confer vi
ABI
lity to 3-deoxy-D-manno-oct-2-ulosonic acid-depleted
Escherichia coli
,
Molecular Micro
BIOLOG
y
, 2008,
67(3)
, 633–648

Meredith, et al, Redefining the Requisite Lipopolysaccharide Structure in
Escherichia coli
,
ACS Chemical
BIOLOG
y
, 2006,
1(1)
, 33-42

Brandenburg, et al, The Expression of Endotoxic Activity in the Limulus Test as Compared to Cytokine Production in Immune Cells,
Current Medicinal Chemistry
, 2009,
16,
2653-2660

Gutsmann, et al. Structural prerequisites for endotoxic activity in the Limulus test as compared to cytokine production in mononuclear cells,
Innate Immunity
, 2010,
16(1)
, 39-47

Beom Seok Park1 et al., The structural basis of lipopolysaccharide recognition by the TLR4–MD-2 complex,
Nature
458
, 1191-1195 (30 April 2009)


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ClearColi Licensing Information:

ClearColi? Competent cells are subject to US Patent 8,303,964 and other US and foreign pending patents.


Lucigen
Corporation ("
Lucigen
") has a license from Research Corporation Technologies to sell ClearColi competent cells to third-parties for non-commercial research purposes only. A separate license is required for any commercial use. For more information about the use of this product by commercial entities, please review our?full licensing page.

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ORDER INFORMATION
Each?ClearColi? BL21(DE3) Electocompetent Cell Kit contains: ClearColi BL21(DE3) Electocompetent Cells in DUO packaging (2 transformations per tube), Expression Recovery Medium (lactose minus), pUC19 Positive Control Plasmid, and complete protocols.

Expression Recovery Medium (lactose minus) is also available separately.