Q: What is the difference between lipid IVA and LPS?
A: Lipid IVA is a modified LPS molecule that does not induce an endotoxic response in human cells. The six acyl chains of LPS (Figure 1) that trigger the endotoxic response are recognized by Toll-like receptor 4 (TLR4) in complex with myeloid differentiation factor 2 (MD-2), causing NF-κB activation and production of proinflammatory cytokines. Lipid IVA lacks the two secondary acyl chains and does not induce formation of the activated hTLR4/MD-2 complex, thus evading the endotoxic response. In addition, the oligosaccharide chain is deleted, making it easier to remove the resulting homogenous lipid IVA from any downstream product.
Figure 1: Comparison of ClearColi lipid IVA and LPS from unmodified E. coli. In ClearColi, two of the six acyl chains have been removed to disable the endotoxin signal, and the oligosaccharide chain has been deleted.
Q: Are ClearColi cells really “endotoxin free”?
A: ClearColi cells contain Lipid IVA instead of LPS. Lipid IVA is incapable of inducing an endotoxic response in human immune cells, but may act as an endotoxic activator in other mammalian hosts such as mouse, Chinese hamster or equine cells. Recognition and immune-stimulatory activity of lipid IVA is affected by species-specific differences in the structures of TLR4 and MD-2. Other species like pigs and rabbits have not yet been tested in our labs.
However, with proper controls, plasmids and proteins can be produced from ClearColi cells without need for downstream endotoxin removal steps. In our experience lipid IVA is much easier to remove from products than LPS. As a result, essentially all of the lipid IVA can be removed from protein products expressed in the ClearColi system, resulting in a protein with minimal endotoxic activity.
Q: Why does the LAL endotoxin assay give a false positive with proteins produced from ClearColi?
A: The Limulus amebocyte lysate (LAL) assay is a common, FDA-approved method for endotoxin detection. The LAL assay and recombinant versions of the assay react with lipid IVA through the 4´-monophosphoryl-diglucosamine backbone structure present in both LPS and lipid IVA. For this reason, the LAL assay is not effective for discriminating between LPS and lipid IVA. However, the LAL assay is an effective assay for identifying lipid IVA impurities in your product.
The LAL assay is also not effective at predicting endotoxicity of lipid IVA. The lipid structural elements recognized by the LAL assay are different from those necessary for immune cell stimulation. Even though the LAL assay reacts with both LPS and lipid IVA, LPS is endotoxically active and lipid IVA is endotoxically inactive in humans. We suggest that users consider alternative physiologically relevant assays.
Q: What other endotoxin assays have you tested?
A: We have used a number of assays to clearly distinguish between LPS and lipid IVA, and to confirm that proteins and extracts containing lipid IVA do not stimulate endotoxic effects in human cells. We have shown negligible NF-kB induction occurs in HEK-BlueTM hTLR4/MD-2 cells even when exposed to substantial concentrations of lipid IVA (Figure 2).
Figure 2: Comparison of relative NF-κB induction in HEK-BlueTM Cells using LPS purified from a K-12 E. coli strain, or synthetically manufactured Lipid IVA.
Human macrophages are sensitive to endotoxin at picomolar concentrations. When stimulated, macrophages release various cytokines including TNFα. Substantial concentrations of ClearColi membrane extracts failed to stimulate human macrophages.
Q: What are the growth characteristics of ClearColi BL21(DE3) cells?
A: ClearColi BL21(DE3) cells initially grow at approximately 50% of the rate of unmodified BL21(DE3) cells, on plates and in liquid media. Very small colonies are visible within the first 24 hours after plating transformants. Some users see variation in colony size; a small portion (<2%) of colonies may be larger than the general population. In general these larger colonies have shown protein expression and endotoxin levels similar to the typical size colonies.
ClearColi cells initially exhibit slower growth rates in liquid culture compared to common BL21 (DE3) strains (Figure 4), but after the initial growth lag, the growth rate and doubling times in liquid culture closely matched that of BL21 (DE3), reaching densities appropriate for protein induction within 3-4 hours after dilution to OD600 of 0.1.
For best results:
Figure 3: Comparison of ClearColi BL21(DE3) growth rate to that of standard BL21(DE3). ClearColi cells experience an initial growth lag compared to unmodified BL21(DE3). After this lag time, growth rates are similar (hours 3-6).
Q: What kind of media should I use for ClearColi?
Because ClearColi is osmosensitive, it is important to include salt concentrations of at least 10 g NaCl/L. Use high-salt LB-Miller (10 g NaCl/L) media for both agar plates and liquid media to maintain higher growth rates. Common media additives such as glucose and Mg2+ may increase growth rates in some cases. Avoid sources of Ca2+, which slows the growth rate of ClearColi. See our Application Note on ClearColi Media for more information.
Q: Is there a simple method to confirm the ClearColi phenotype?
A: Yes, due to the modified outer membrane, the ClearColi strains are sensitive to a number of agents including bile salts. A simple method to test for the phenotype of the ClearColi strains is to grow the cells on the following set of plates:
Q: Given the modified membrane structure, are there any differences in the cell membrane integrity?
A: In our experience the mechanical cell membrane integrity of ClearColi strains is similar to that of wild-type strains when subject to sheer forces. The modified membrane structure does give the strains a number of different characteristics. Due to the lack of any LPS glycosylation, the cell surface is more hydrophobic than the wild-type strains, which makes ClearColi more likely to aggregate and settle out of solution. The cell wall also seems to have greater permeability, rendering the cells more osmosensitive.
For best results:
Q: What levels of protein expression can I expect from ClearColi BL21(DE3) cells?
A: The ClearColi system has been used by our customers to express several dozen unique proteins. See our citations for a few examples. In general the recombinant protein yields for the ClearColi system have been reported to be similar to those produced from other commercially available BL21(DE3) strains. Some variation of expression levels may be seen with different proteins. However, if ClearColi produces protein yields substantially lower than yields from other BL21(DE3) strains we recommend the following steps:
Q: Can other non-T7 induction protocols be used?
A: We have not tested alternative induction protocols. However, we expect that induction protocols and promoters used with other BL21(DE3) strains should also work with ClearColi strains.
Q: Are recombinant proteins expressed from ClearColi BL21(DE3) cells soluble and functional?
A: Lucigen has expressed soluble, functional fluorescent proteins from ClearColi BL21(DE3) cells. No significant difference in protein solubility or functionality is expected between unmodified BL21(DE3) and ClearColi BL21(DE3) cells.
Q: Have you produced proteins at scale?
A: Our customers have reported growth of ClearColi strains in a 5 liter fermenter and shown optical density levels in excess of 150, and that some defoaming agents (particularly silicone-based defoamers) are beneficial while other defoaming agents such as polypropylene glycol may inhibit cell growth.
Q: What is the best way to purify my protein?
A: We and our customers have used immobilized metal ion affinity chromatography (IMAC) and size exclusion chromatography (SEC) to purify proteins of interest. Lipid IVA is a homogenous molecule comprising a specific molecular mass and charge making standard purification techniques more effective at removing lipid IVA than LPS. However, a general protein purification protocol does not exist, not even when ClearColi strains are being used for protein expression. Just like with any protein expression platform, the purification scheme largely depends on the specific properties of the protein of interest and must be established in each particular case considering the protein´s physico-chemical properties, size, biological activity, binding affinity etc.
Q: Is endotoxin removal after purification necessary?
A: The answer depends on the user's method of endotoxin measurement and the application. As previously discussed, the lipid IVA of ClearColi cells does not cause an endotoxic response in human cells; however, LAL testing may result in a false positive result.
A simple Ni-column purification step for proteins produced from ClearColi cells significantly reduces LAL response levels. For example, Lucigen demonstrated a 99% reduction in LAL response comparing ApoA1 produced in ClearColi cells versus E. cloni® EXPRESS BL21(DE3) Electrocompetent Cells. See our application note in Nature Methods. ApoA1 is known to bind LPS, and as a result is notoriously difficult to purify.
LAL Results (EU/mg)
ClearColi® Electrocompetent Cells
E. cloni® EXPRESS BL21(DE3) Electrocompetent Cells
The lack of lipid IVA in the protein preparation, as measured by the LAL assay, varies from protein to protein. Proteins known to bind LPS may have higher residual lipid IVA after minimal purification. Other proteins have demonstrated LAL results of less than 1 EU/mg.
In applications where minimal endotoxin levels are critical, additional cleanup steps should be taken. There are also a number of cell-wall components such as peptidoglycan that may activate the immune system. Purification is necessary to remove these other cell-wall components.
Q: How do I avoid contaminating my proteins or plasmids with endogenous LPS?
A: Good laboratory sterile technique can adequately control LPS contamination from extraneous sources. Lucigen recommends the following precautions.
Q: Do I need a license to use the ClearColi system?
A: Users receive a limited use license with the purchase of a ClearColi kit. This license allows evaluation of the ClearColi system for non-commercial purposes. Academic users may use the ClearColi system under the limited use license for non-commercial use; no further license agreement is required. Academic laboratories may not use the ClearColi system for commercial purposes or transfer ClearColi materials without RCT’s prior written permission. RCT will work with you to accommodate material transfers.
For commercial entities and non-profit entities engaged in commercial activities, the limited use license automatically terminates twelve months after purchase, and users are required to take a ClearColi commercial research or product license. Commercial research licenses allow use of ClearColi for internal research purposes. Product licenses allow production and sale of product made using the ClearColi system. For additional details please contact firstname.lastname@example.org.