First-check the Q&A forums there are hundreds of similar threads on ResearchGate that that have already been answered very well!

Is your protease really cleaving itself from the GST or are you just seeing truncated forms due to poor E.coli ribosome performance? There are plenty of serine protease inhibitors but take care as the most common (PMSF/AEBSF) will irreversibly inhibit your protease activity! I am assuming that you want to work with your protease in an active form.

Then..

As you already have a construct that expresses then there are a few things that you can try (I am assuming you are using T7, LB and IPTG at 37°C so far with a globular protein and not an IDP).

Firstly if you want to continue to use IPTG for induction use a richer, buffered medium , terrific broth is simple to make but can improve soluble yields dramatically over LB.

Try reducing temperatures post-induction to 16-20°

Try reducing IPTG concentration-although in most e.coli expression strains induction is not really titratable.

Try auto-induction media instead of IPTG

If none of these work then think of changing your fusion protein or tag, I particularly like SUMO, thioredoxin and Z-tag (and much prefer them to GST and MBP) and/or changing the boundaries of your construct to avoid e.g. unstructured domains. There is no sure way of getting soluble protein that stays soluble after cleavage from the fusion but these should help.

Of course the effects of all of these are dependent on your protein and it may be that you ultimately have to accept re-folding from inclusion bodies.

Good Luck

P.S this answer is a copy of my answer to a similar older thread

 First-check the Q&A forums there are hundreds of similar threads on ResearchGate that that have already been answered very well!

Is your protease really cleaving itself from the GST or are you just seeing truncated forms due to poor E.coli ribosome performance? There are plenty of serine protease inhibitors but take care as the most common (PMSF/AEBSF) will irreversibly inhibit your protease activity! I am assuming that you want to work with your protease in an active form.

Then..

As you already have a construct that expresses then there are a few things that you can try (I am assuming you are using T7, LB and IPTG at 37°C so far with a globular protein and not an IDP).

Firstly if you want to continue to use IPTG for induction use a richer, buffered medium , terrific broth is simple to make but can improve soluble yields dramatically over LB.

Try reducing temperatures post-induction to 16-20°

Try reducing IPTG concentration-although in most e.coli expression strains induction is not really titratable.

Try auto-induction media instead of IPTG

If none of these work then think of changing your fusion protein or tag, I particularly like SUMO, thioredoxin and Z-tag (and much prefer them to GST and MBP) and/or changing the boundaries of your construct to avoid e.g. unstructured domains. There is no sure way of getting soluble protein that stays soluble after cleavage from the fusion but these should help.

Of course the effects of all of these are dependent on your protein and it may be that you ultimately have to accept re-folding from inclusion bodies.

Good Luck

P.S this answer is a copy of my answer to a similar older thread

Hi Chitra, just to add to Nick's answer, after induction, find the optimal time point in which the proteinase is soluble before it gets deposited into inclusion bodies.

Proteins in inclusion bodies are a mess of denatured proteins that are rarely usable for anything. 

Also, what is the substrate specificity of this serine proteinase? You might be able to do a simple affinity isolation of this proteinase after a short induction. 

Hi there,

is proteolysis occuring at one single site on your fusion protein. If it's the case it should be worth considering mutation to alter this site and make it insensitive to your protease.

Dear Chitra

I had similar problems expressing one serine protease from Bacillus. Finally I decided to purify the protease from inclusion bodies. Resulted to be a quite pure fraction, and it prevented proteolysis problems. After that I refolded the protease in vitro. But of course, it will depend on the final application for your protease. I think Nick answered the most common ways to improve solubility.

Best regards.

Jose...I want to use it for crystallization so refolding is the last thing I would resort to.

Nick... Yes, its getting cleaved from GST. I have already tried His tag but in vain.

Depending on the cleavage site (that you could characterize by n-term sequencing or MS of the fragments)

-if it is in your protein you could try Dominique's suggestion-mutating the site or even starting your fusion protein after this site (later in your protease sequence) if the activity of your enzyme is unaffected by an N-term truncation.

-or if it is in the c-term of GST or the linker/thrombin site you could try out something different like a SUMO fusion (no linker and different cleavage site etc. and is also very good at solubilizing proteins.

Is a his-tag version also getting cleaved or it is just insoluble?

-if it is cleaved then it is more likely that the site is in your protease sequence

-if it is just insoluble then you could give some of the other suggestions a go to improve the solubility.

I have tried SUMO and His tag as well but protein remains insoluble in both the cases.