Dear Paratsou

From the gel it seems that you have a protein degradation issue (because you have a >45KDa in the insoluble and 14 in the soluble which together may correspond yo your full length protein.

Which was you induction temperature? 37°C

In this case you can certainly go down to 25°C or 20°C to see if in this way you may reduce the degradation. (proteases are less active at low temperature)

Are you expressing the protein with some tag? and the fragmentation may be localized between protein and ta (in the linker?) or it seems to be localized inside the protein sequence?

Because in the first case you can try to change the linker sequence, while in the last case you have to identify the digestion site (eg performing N-terminal sequence analysis by EDMAN approach) and if possibile try to remove the digestion site performing single point mutations.

One other approach could be, change the E.coli strain or expression hosts.

For example in the past, for the catalitic domain of TcdA clostidium that i was never able to express in E.coli, because we obtain in insoluble and fragmented form, we moved in Brevibacillus and we were able to express it in folded and active form.

Article The structure of Clostridium difficile toxin A glucosyltrans...

but you have to try many things in E.coli before move on one other organism.

good luck

Manuele

Dear Paratsou

From the gel it seems that you have a protein degradation issue (because you have a >45KDa in the insoluble and 14 in the soluble which together may correspond yo your full length protein.

Which was you induction temperature? 37°C

In this case you can certainly go down to 25°C or 20°C to see if in this way you may reduce the degradation. (proteases are less active at low temperature)

Are you expressing the protein with some tag? and the fragmentation may be localized between protein and ta (in the linker?) or it seems to be localized inside the protein sequence?

Because in the first case you can try to change the linker sequence, while in the last case you have to identify the digestion site (eg performing N-terminal sequence analysis by EDMAN approach) and if possibile try to remove the digestion site performing single point mutations.

One other approach could be, change the E.coli strain or expression hosts.

For example in the past, for the catalitic domain of TcdA clostidium that i was never able to express in E.coli, because we obtain in insoluble and fragmented form, we moved in Brevibacillus and we were able to express it in folded and active form.

Article The structure of Clostridium difficile toxin A glucosyltrans...

but you have to try many things in E.coli before move on one other organism.

good luck

Manuele

Dear old friend, Parastu,

Check your protein in its original form, it may be a glycosylated protein to reach 66 kDa, and your expressed one is not glycated (due to the expression host, BL21)

Check once more your gene, I had a similar experience in which my protein had a single base error and made a stop codon, therefore the truncated form was produced.

I see a band around 15-16kDa in the soluble fractions at both temperatures. This band doesn't seem to show up in the whole cell or before induction lanes. It seems almost proportional in intensity to the induced protein. If you add this 15-16kDa portion to the 46kDa band, it almost makes up the difference to your full length protein. So, there is a possiblity that the protein is getting cleaved upon synthesis. Have you tagged the protein with something that is 15-16kDa ?

Else, Mohsen Akbarian 's answer seems viable. You may need to recheck the molecular weight from the mRNA translated FASTA sequence.

I just read @Manuele Martinelli's answer. I agree with him.

Hi Parastou

In addition to the detailed suggestions by others here, I'd like to add that you can try using protease inhibitor cocktails (if you aren't already using them) and lower the induction temperature if you are using 37 degrees currently (as suggested by Manuele). You can then compare the result with what you have shown here. This may help you know whether you are losing your protein to proteases.

All the best

Gayatri

As Mohsen already mentioned, 66kDa protein could be the post translationally modified one and your 45kDa is the non modified.

Thank you very much for your great suggestion.

I should mention somethings here,

dear Manuele Martinelli, I checked two post-induction temperatures, 25 and 43. although I know about increased chance of protein aggregation at high temperatures, I just wanted to check the possibility of higher expression at the elevated temperature.

I have His-Tag at C-Ter of the protein. my protein is homotrimer which fused to a coil sequence (~6.5 KDa).

I can see a protein band around 20 KDa after induction too. I think proteolytic degradation happened which it's also visible at 25 ° C.

Dear Mohsen Akbarian Thanks a lot for your comment. I make sure about the sequencing of the gene and I didn't see any stop codon inside the gene.

this protein has 65.5 KDa molecular weight without any post-translational modification.

Praveesh Valissery Thanks for your answer. as I mentioned, the fused gene to the sequence of my protein has just around ~6.5 KDa molecular weight.

Gayatri Bagree you are right. If proteolytic cleavage happened after harvest, I shouldn't see two separate bands equal my protein MW in the whole-cell analysis.

I mean this proteolytic cleavage happened inside the cell during protein expression because the protein doesn't fold correctly in the right conformation. I should check it in the presence of a chaperone to be sure about that.

While you work on this problem, you can parallely transform your contruct into another strain of bacteria. Like Rosetta-gami or codon optimized BL21. In case you get lucky and this happens to be a host issue. When i get stuck on protein expression/purification problem, I use the Arctic Express DE3 strain. It allows for induction and expression at 11C. So that should take care of folding issues. And the low temperature should reduce any proteolytic cleavage during expression. I usually get my protein properly in the soluble fraction, albeit the amount is less compared to other easy-to-express proteins. And this strain should be common enough that if you dont have it in your lab, a lab in your building might have it.