E. coli strains are good working horses for expressions of recombinant proteins. They have got huge potential for production of low-cost subunit vaccine candidates against various target diseases. However, the presence of E.coli derived endotoxins in the formulation, and expensive cost for their removal from the formulation has become one of the challenges in the bioprocessing of vaccine candidates. Thus, is it possible to engineer E. coli strains that do not produce endotoxins? Did anyone come across such strains?
Short answer: No.
Longer answer:
Lipid A is often what's meant when people say "endotoxin." It's an essential component of gram negative cell membranes and the bacteria will die without it. It's extremely well conserved across species. That's probably why it gets such a strong immune response in the first place. I believe people have engineered E. coli strains to produce less immunogenic versions of Lipid A, but with our current knowledge it's probably not possible to get rid of it completely, and is definitely not as simple as knocking out a couple genes.
Dear Alexandra,
Many thanks for your scientific explanation. I can imagine the challenges. I agree with your point. "It is probably not possible to get rid of lipid A completely". It will be very tricky to knock out all of the critical genes involved in the biosynthesis of bacterial endotoxins. However, I am still wondering the necessity of endotoxins for the survival of gram negative bacteria? There are bacterial cells, such as gram positive ones, surviving without endotoxins.
Hi Alemu, I fully agree with Alexandra. Lipopolysaccharides are fundamental constituents of all bacteria and it is a pipe dream to try to eliminate them for more cost effective vaccines.
Even if you were able to produce a strain of some bacteria that don't have lipopolysaccharides, you still have the problem of bacterial proteins with N-formyl methionine that drives the inflammatory system nuts and will do more harm than good.
Hi, I use Clear Coli from Lucigen, they have modified lipid A and much less endotoxi. Have a look on its web page.
Dear Steingrimur,
Dear Rosa,
Many thanks for your answers. I appreciate it!
Alemu: The reason gram positive bacteria can survive without endotoxins is they have a completely different membrane physiology. Their thick, externally exposed cell wall is able to help protect their inner membrane from rupturing. Even then, they have outer structures like lipoteichoic acid(LTA) which seem to serve a similar purpose to LPS and also activate an immune response. A big job of the LPS in gram negatives is to stabilize the membrane.
Dear Alexandra
Many thanks for your scientific explanations. I appreciate it!
Hi Alemu
Scientist discovered whole sequence of E.coli for many years, thus now we know the genetic sequence that produce endotoxine in this bacteria. therefor we can knock out that sequence with genetic engineering tools.
good luck
Dear Hosein,
Many thanks for your answer. I appreciate it!
Yes, it may be possible to genetic engineering tools or synthetic biology approaches to do that. However, the bacteria may not survive without endotoxins. Have you read answers given by Alexandra Johnson?
The real problem here is that your protein of interest is not necessarily secreted by E.coli (or any bacteria), and most likely you first have to destroy the bacteria to gain access. This is where the endotoxins, be it LPS or lipid A or anything else, become part of your contaminants. If you cannot be bothered to extensive purification steps, you may find it more cost-effective to switch over to a (mammalian) cell line for production. Isolation from culture media might be cheaper and definitely free from endotoxins.
Dear Jan,
Many thanks for your answer and your recommendation. However, high level of purity is desired for human vaccine candidates. Bye the way, is it possible to get E. coli strains secreting recombinant proteins to the extracellular environment (or medium)?
You need high yield as well. You need a system that produces and secrets large amounts in the growth media that can be easily retrieved without destruction of the producing cells. Depending on the molecules you are after, either bacteria or eukaryotic cell line would be best. Don't rule things out upfront. Good luck!
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