It is difficult to get a universal protocol that works for all proteins. I personally prefer to apply a high-throughput screening platform to identify conditions for extraction and purification of individual proteins. You can use a factorial design to test many conditions at a time. Here is the link for the paper if it gives you an idea to develop a suitable protocol for extraction of your target proteins.

https://link.springer.com/article/10.1007/s10295-013-1307-1

Dear Alemu Tekewe,

Thanks for your answer. Generally I want to know is osmotic shock applicable to pLysS strains or not? If yes, I will optimize it. 

Dear Mansoureh 

You can certanilly try to apply a standard osmotic shock protocoll for the extraction of the protein from it. You may risk to lise some cell completly and reduce a little the protein purify but i don think this will represent a main issue.

in the same manual they told:

"When produced from the cloned gene, relatively high levels of T7 lysozyme can be tolerated by E. coli, apparently because the protein is unable to pass though the inner membrane to reach the peptidoglycan layer"

so it not seems that the T7 lysozime for them cause the distrption of the inner membrane, but that inner membrane prevent the lysozime release from the cells.

However, why are you using plyss strain? 

Is you protein toxic and you have problem of cell growth before induction?

In my experience in very few cases, using a media containing glucose, able to repress itself the expression of T7 promoter, plyss perfomed better than standard BL21(DE3).

good luck.

Manuele

Dear Manuele, 

Thank you very much for your answer. 

Hi Mansoureh,

I'm just wondering how your problem got solved. Because I'm trying to express alpha-synuclein in E.coli and it's been proven to mainly express in periplasm. My protocol used BL21(DE3) and showed a huge success but I only have BL21(DE3) plysS strain and failed. So I'm thinking what you said might be the reason to account for it? Have you had any success using plysS for osmotic shock? Thanks!

Peng

Dear peng,

I have used osmotic shock protocol in noth Bl21(DE3) and PlysS. In the periplasmic fraction of both strains, I could see my protein. But the size of extracted protein was larger than the native protein used as control (in both SDS_PAGE and Western_blot). It means that the pelB signal peptide was not cleaved from the protein. May be the pelB was not suitable for my protein or may be the extracted protein was from cytoplasmic fraction and released as the result of complete lysis.

All of the above are my guesses. I did not have enough time to test anothe SP, so I ordered cytoplasmic construct (without SP) and continued.

Dear peng,

I have used osmotic shock protocol in both Bl21(DE3) and PlysS. In the periplasmic fraction of both strains, I could see my protein. But the size of extracted protein was larger than the native protein used as control (in both SDS_PAGE and Western_blot). It means that the pelB signal peptide was not cleaved from the protein. May be the pelB was not suitable for my protein or may be the extracted protein was from cytoplasmic fraction and released as the result of complete lysis.

All of the above are my guesses. I did not have enough time to test another SP, so I ordered cytoplasmic construct (without SP) and continued.