Hi Joshua,

For Cell Lysis:

I freeze thaw my pellets around 4-5 times -80 & 42C,

Add lysis buffer/lysozyme/PMSF

Sonication on ICE (for 5 mins: 15 seconds on & pause at 3) about 6 times for the sample untill its viscosity is less dense

sometimes I fail to control froth formation but glycerol 30% addition helped in minimizing this froth

Then centrifuge at 6200 rpm 4C for 20 mins and separate the supernatant from the pellet.

This is how I do it

Thats how it is! I am sure you are trying to make a Human/non-bacterial protein in bacteria? Nevertheless, you still have options to use other constructs to make GST/MBP/Trx/SUMO fusion proteins which are known to enhance the solubility.

Good luck!

No No it's NOT a non-bacterial protein..it's a bacterial one!! And Yes I tried the petSumo system , am actually working with both expression vectors but thought since the sumo fusion protein is quite big ,made my protein KDa jumps from 36 to 51kDa that may affect its folding , that's why it's in inclusion bodies as well, I'll keep trying....Thanks For your help :)

Hi Hadeel, using different e.coli strains for expression might help. If you still have the cDNA for your protein of interest with the original codons you could try BL21 (DE3) Rosetta. This e.coli strain carries tRNAs with human codons to get a better codon usage in e.coli. Sometimes the codon usage can influence the proper folding of proteins a lot. E.coli BL21(DE3) Origmai carry chaperones for better folding.

Since you mentioned that you already see inclusion bodies before induction, another option to avoid "leaky expression" before induction is to add 1% (w/v) glucose to your culture medium to keep the lac-promotor quiet before induction. Also use only short induction times at 30C or lower. If this doesn't help switching to other tags like Ananda suggested would also be an option. MBP make nearly every protein soluble.

If your protein contains several methionins/cycteins and you have access to isotope lab you can also try 35S in vitro translation, but this yields only little amounts of protein. But because of the high sensitivity of radioactiv labeling pull-downs e.g. can be performed.

The position of the His-tag can also influence expression. So you might try c-terminal His-tag

Just to be sure: Did you check for proper lysis of your e.coli under a microscope? If your cells are not properly lysed your protein will appear in the pellet as well.

Best,

Kai

Hello Kai, I guess I will give a try for 1 % (w/v) glucose, also with uninduced & Empty plasmid cultures as controls just to be comfortable with the results.

For the his-tag positioning:

I am using the pet28b which is a C-terminal His-tag ,while the petSumo is N-terminal & both gave me inclusion bodies!!.I see my options for changing the strains..

For Cell lysis:

you mentioned examining under microscope, I never did that...How would it look like after proper sonication..I guess more likely disrupted shapes & no intact cell shape,right??

Thanks alot for your help Kai :)

Hi Hadeel, If you check your cells for proper disruption under microscope you should compare sonified vs. untreated cells. You will see the difference! You will only have small vesicles left if the sonication worked well. Another point is that you really have to take care that you do not heat your lysate during sonication. Always use several short treatments on ice-water and let lysate cool down after each sonication round, since the sonotrode will heat up your lysate pretty much.

Best,

Kai

Hi Hadeel,

maybe induction is not the problem.... but the "solubilisation" after bacteria disruption is....

How do you know that your protein forms inclusion bodies ???

is it just because you see it in your pellet fraction ???

If your protein is insoluble:

Perhaps, instead of trying to play with induction conditions, you should try to fix one induction condition (eg 4h @ 16C in the presnece of 0.1mM IPTG)

and work out which lysis buffer component you could change to increase the solubility.

If your protein is in the inclusion bodies in vivo:

If your protein are forming inclusion bodies and that you do not want/can purify your protein using denaturating condition (eg Urea 8M on the inclusion bodies contained in the pellet fraction) you should think about changing your tags or use fusion as MBP etc... to try to increase the solubility in vivo...

hope that help a bit

good luck

Nico

Hello Nicolas,

Yes I see my protein in the pellet fraction versus almost nothing in the supernatant , For the lysis buffer : I tried adding 30% glycerol to increase solubility ,it worked but still very faint band in supernatant ,Do you know other additives that could improve solubility but not denaturating?

Thanks alot !!

you can increase the NaCl concentration

or change to KCl

or/and add detergents (depends on your protein)

In addition, if the glycerol changed the solubility

you are on the right way....

then what you should really do is to try to optimize the lysis buffer and not to try to change the inducing conditions

Thanks alot Nicolas for your advice & I hope am on the right track :)!!!

How are you lysing the cells? Often proteins that appear in the pellet can be due to incomplete lyses.

Hi Joshua,

For Cell Lysis:

I freeze thaw my pellets around 4-5 times -80 & 42C,

Add lysis buffer/lysozyme/PMSF

Sonication on ICE (for 5 mins: 15 seconds on & pause at 3) about 6 times for the sample untill its viscosity is less dense

sometimes I fail to control froth formation but glycerol 30% addition helped in minimizing this froth

Then centrifuge at 6200 rpm 4C for 20 mins and separate the supernatant from the pellet.

This is how I do it

Hi Hendeel,

There could be several problems on your experiment. Do you know if your protein are happy by itself? Or it might need another protein to coexpress to make it more soluble. How much NaCl do you add into your lysis buffer? I would try to increase the [NaCl] up to 300 mM in the lysis. Then, try to break open cells again.

For cell lysis, I think you should try a milder condition to lyse bacterial cells. I use lysozyme to digest the cell wall at RT for 30 minutes. Then, sonicate for just 3 times: 10 sec pulse , 10 second off, put sample on ice for 1 min, and repeat the sonication again 2 times. This way, I usually get at least 80% cell lysis. If cell lysis is efficient, you should see almost every protein band the E. coli has in the supernatant, not in the pellet.

Is your protein induced strongly or weakly when you use 0.2 mM IPTG at 37C?

With inclusion bodies you always start the fight as you did by the temp. decrease. Than what you do is to make sure enough of the oxygen is available for bacteria, the tricks are:

- no more than 20% volume of the culture in your Erlenmeyer flask (200ml in 1l flask)

- maximum speed in your shaker, with the maximum angle possible

- no tight cover on your flask (punctured aluminum foil, I do it without any cover)

- make sure your bacterial culture is 18-16 degrees before you add IPTG (20min in higher temp. is enough to express protein and start IB formation you can't stop)

Hi Hadel, I am having the same problem with the presence of inclusion bodies, I want to know what strategy did you follow to reduce them.

Thanks

@ hadeel

please elaborate on the methods you followed i am facing same problem

Proteins that form inclusion bodies are a utter pain. If you have a small amount of soluble protein you might be able to swing the balance by optimizing growth. Try cold shock on ice for 20min before adding IPTG (0.4mM or less) induce at 30'C, drop the shaking to 120rpm. This will up chaperone levels, slow the cells metabolism and growth rate down - giving time for you protein to fold! If there really is no soluble protein, try adding a His-tag, GSH or MBP tag this should drag some protein into the soluble fraction. If you have an assay to test the function of your protein you could always denature and refold the material from the inclusion bodies - I suggests using a commercial refolding screen.

I was thinking exactly the same as Jason Crack about changing the tag for example GST, it is probable that this increases the solubility of your protein, especially if it is really important for you to purify under native conditions. The advantage of GST is also quite small molecular weight.

Good luck :)

why not try a weaker promoter, which can slow the expression speed, and might help the folding

Often SUMO tag helps to the problem of solubility!!

Hi, Did you solve your problems. I also get into this situation (inclusion body ). Could you give me some help?