I just quickly checked on the PDB and there are a few crystal structures for human BCL-2 expressed in E. coli.

Maybe this can help:

http://www.ncbi.nlm.nih.gov/pubmed/?term=26858413

http://europepmc.org/articles/PMC30598;jsessionid=Dg7qlVuk8RXASxFT5LFx.0

Dear Lax, 

since the BCL2 protein is bound to the mitochondrial membrane, the classical methods of His-tag purification are not recommended due to the solubility problems. Here I send you a link with a publication where a full- length human BCL2 is purified fro E.coli using a batch-based cell-free expression system.

http://www.sciencedirect.com/science/article/pii/S1046592811000313

I would recommend you to (always) do a small scale purification before you go large scale to be sure that the protocol you choose is going to work.

In any case I have experience for some proteins, in which you do not see a dramatic induction of the expression, but when purifying it, the amount of protein was enough for my applications (EMSA and MST).

All the best

Ingrid

Hi Lax,

Membrane proteins are a specialized case of protein expression and purification. It is very common that they don't express well in E.coli, especially if your protein is supposed to have any PTMs. This is may be why in the paper Ingrid linked to above they've opted for a cell-free system.

To isolate the membrane protein you need to solubilize it - i.e. extricate it from the membrane it's sat in. You can use detergents for this. This is why you can use triton X-100 to visualize it on an SDS gel. 

I wouldn't recommend straight off adding 1% Triton to your lysis buffer, however. I'm not sure what your downstream applications are, but you say you're doing large-scale production. So, potentially crystallization? Some detergents are much harsher on the protein than others and won't be good if you want to crystallize the protein. Triton is quite harsh. Others, such as DM or DDM (as random examples) may be a better bet. They are expensive however, and you would need to take the time to screen different detergents to see which works best for your protein and system.

I hope this is of help to you!

Good luck,

James

Dear,

It problem of over expression. So for any protein First of all standardize expression conditions so you can avoid inclusion body (Condition combinations- Time of expression 2-15 hrs , temp 15, 37 degrees, IPTG 0.1 to 1 mM)  and see which condition gives you good expression in supernatant. Please avoid to get protein from inclusion body. You will waste your time. For best follow protocol " Protein Expression. A Practical Approach by Higgins'  For this you require 3 days. If your protein is membrane protein this approach do not work.

First - pH of buffer  is important 

second- do not use single colony use 20 to 25 colonies.Do not worry all are from pur plasmid preparation only.

Regards.

Rajvardhan