Sometimes, overexpressing a biologically active protein in its native state is toxic to the cells. Sometimes, the amounts of active, soluble material produced are minuscule. Sometimes it is just easier to purify and refold the protein from bacterial inclusion bodies than to start from large-scale fermenter-loads of mammalian or insect cells. Since bacteria have a reduced repertoire of post-translational modifications, bacterial production may yield more homogenious material (e.g. concerning phosphorylation, glycosylation) Each protein is different - since bacterial production. If refolding from inclusion bodies works, it may be the fastest and cheapest method to produce large amounts of material. If it does not work, you can still progress to more laborious and more expensive methods.

Cause they are biologists that know nothing about physics 

They just follow a recipe

Sometimes, overexpressing a biologically active protein in its native state is toxic to the cells. Sometimes, the amounts of active, soluble material produced are minuscule. Sometimes it is just easier to purify and refold the protein from bacterial inclusion bodies than to start from large-scale fermenter-loads of mammalian or insect cells. Since bacteria have a reduced repertoire of post-translational modifications, bacterial production may yield more homogenious material (e.g. concerning phosphorylation, glycosylation) Each protein is different - since bacterial production. If refolding from inclusion bodies works, it may be the fastest and cheapest method to produce large amounts of material. If it does not work, you can still progress to more laborious and more expensive methods.

Hi Goran, from my postdoc days, my boss was trying to produce plasminogen activator-1 (PAI-1) from E. coli. Pai-1 is a conformationally sensitive serine proteinase inhibitor. 

He was able to isolate gram quantities of it from inclusion bodies. but it was a jumbled mess of proteins, and a small % could only work upon urea/Guanidinium denaturation. The rest of the PAI-1 just precipitated and lost.

He then hit up on the idea of catching the soluble PAI-1 in E. coli after inducement but before it got deposited into inclusion bodies.

After around 3 hours after IPTG (if I remember correctly), the bugs were lysed. The yields were 10% compared to the inclusion bodies, but all the PAI-1 was soluble and could be recovered intact without denaturation/renaturation. 

I think I understand what Steingrimur says, you should do anything you can to keep the production of  soluble recombinant protein. 

Hi Annemarie, there is no universal induction/purification protocol that can be applied to all proteins all of the time.

What I am trying to bring attention in my question is that a lot of researchers are just following a protocol without asking questions.

Protocol says, you induce overnight.

No problem. I induce overnight.

Protocol says to lyse bacteria and centrifuge out inclusion bodies containing my protein(s) of interest. 

No problem, I lyse the bacteria and isolate the inclusion bodies.

I have a big pellet in my centrifugation tube.

Somewhere in there is my protein, but I have to solubilize the pellet and purify it! 

This where most of the problems come in. Many researchers regard protein as nails that can be fixed or put into place with a hammer.

You can solubilize a protein, be it with it urea, sds, Guanidinium, etc, (e.g hammers),  but they still dont fit.

If I can get that idea across hat you should not use inclusion body proteins, I'm happy.

It's all about finding the right recombinant production host for your target protein... Is it a intracellular protein or a secreted one? is it prokaryotic or eukaryotic? does it get post-translation modifications such as glycosylation, proteolytic processing, etc? Without a due consideration of these basic factors, one can end up with inclusion bodies, such as in simple recombinant host such as E. coli, but then again, depending on the properties of the native protein, even the inclusion bodies would not be the end of the road, and may instead offer an advantage during the purification of the recombinant product... 

@David. Thank you for your contribution. That's the case I was trying to bring attention to.

There are plenty of options to isolate proteins before they go into this mess that constitute inclusion bodies.

Hi everybody and thank you for your contributions.

I started this thread because A LOT of questions posted on RG involve how to solubilize proteins deposited into inclusion bodies. It frustrated me.

I answered many of them, trying to have the researchers see that there are simple ways of harvesting proteins before they aggregate and are almost impossible to recover intact.

I think Omar is correct in that many junior researchers just blindly follow protocols and are flummoxed when they end up with a dense pellet of insoluble protein crap.  

Thanks Steingrimur, I was referring exactly to that. If some people got offended, I am still waiting for their arguments. As Steingrimur says it amazes me the number of researchers that do not even know what an inclusion body is or how it was formed. 

Hi David, I expanded the topics on RG where this thread will be seen. 

I Hope it helps.

Hi all,

I'm a big fan of inclusion bodies. You get the highest titers from E coli from IBs (10 g/L broth is not uncommon and I've seen higher compared to 1-3 g/L soluble- either periplasmic or intracellular, the latter a little higher), and refolding is really not that difficult. It's an extra step to solubilize and refold, but you can start with 80+% pure protein from IBs and obtain 60-100% refold efficiency of active protein. They're a great intermediate storage form as well.  Several protocols are available in the literature and you will have to find the best one for your protein, but it's worth it.

@Omar How is physics related to inclusion bodies or protein expression?

Hi Denis, if protein isolation from inclusion bodies works for your research then good for you!

I'm hoping that other researchers will evaluate the pros and cons of isolating proteins from inclusion bodies.

I also wonder why almost everyone is still using IPTG, although lactose (auto-)induction almost always gives better (or at least the same) yields and it's cheaper and less toxic to cells.

Lactose has to be converted to allo-lactose by the cell first and is also hydrolyzable by B-galactosidase, and can function as both an inducer and carbon source.  This may not be desirable whereas IPTG is non-hydrolyzable. The main reason is many cheap sources of lactose are derived from animal sources (BSE/TSE) which disqualifies it from use in production of human biopharmaceuticals. 

"The main reason is many cheap sources of lactose are derived from animal sources (BSE/TSE) which disqualifies it from use in production of human biopharmaceuticals."

The USP grade lactose (from milk) is cheap and - since it's USP - I suppose it can be used for biopharmaceutical purposes.

Anyway, my impressions are from the academic environment where this is not an issue.

@Michael

do you have any support for your claims regarding lactose?

BTW IPTG can be used for autoinduction as well.

These are not 'claims', these are facts:

http://www.ncbi.nlm.nih.gov/pubmed/15915565

(My personal experience also supports this but I've used it for very few truly different proteins)