Why a previously soluble protein is now going into inclusion bodies? - protein expression (Jun/07/2008 )

Hi everyone,

I'm new to this forum and am having problems expressing a bacterial cytoplasmic protein in E col BL21(DE3) cells.

I had previously expressed and purified the cyoplasmic domain of a sensor kinase using Ni-NTA affinity matrices and had no problems. I had to make more protein 6 months later and re-transformed the plasmid into my electrocompetent BL21(DE3) cells. I then grew 500ml cultures of cells and induced with 1mM IPTG for 4 hr. However, unlike the previous time where the protein was soluble, most of the protein had gone into inclusion bodies. I thought that it was a problem with my competent cells and prepared a fresh batch but the most protein was in the insoluble fraction.

I have also tried induction with lower IPTG concentrations (0.05 -- 1mM) and lower temperatures (15, 25 degrees) but still get very low amounts of soluble protein.

I don't understand why a protein that had been so easily expressed before is not so anymore, even though all conditions were maintained. The only other difference is
that I made new plasmid.

Have you ever come across a situation when a previously soluble protein became insoluble? I would greatly appreciate any advice.

hdc

Hi

Did you check the expression difference between your earlier host and Bl21 clone. IB formation is mainly based on your expressed Protein and the growth conditions of the host.
And what is the ratio of expression your protein in soluble form and insoluble form, if your expression is more then probably it is going in ib formation.

Try to grow you r culture at low temperatures from the induciton( better prior induction)

all the best

Hi everyone

Thanks for the help and comments.

Compared to previous expression, the amount of of total expressed protein i'm getting now is a less and about 90% is going to inclusion bodies. Expression conditions were the same as before. Lowering the temperature didn't seem to help.

I'm thinking of switching host strain to see whether i can get more soluble protein. I'm going to do a small scale culture to test expression at different temp, diff. IPTG concentrations and take samples at different times. Alternatively, I read that you can express the protein with tags that make the protein soluble (e.g. NusA, thioredoxin) and the tags can later be cleaved of with TEV protease. I might try doing this if switching host does not help.

We'll see how things go...

hdc