Hello, everyone,
I am trying to express a GST taged human protein domain in E. coli. The fragment is cloned into pGEX6p-1 and has correct sequence and reading frame. I have tried many ways including changes of temperature, IPTG concentration and even the host cells ( I tried BL21, BL21(DE3)-RP, BL21(DE3)-RIL). Finding the BL21(DE3)-RP worked out. But most of the objected protein is expressed in inclusion body,the soluble is a little.So I lower the temperature to 27℃.I only get 3mg objected soluble protein from 5 liter culture medium.Could anyone please give me some idea and suggestions how to improve the soluble protein expression?
Thanks a lot.

When this happened to me I switched to Baculovirus expression in SF9 cells and it worked.

Well, you can still try differents OD (0,4-2,0) and a media with glycerol and or glucose.Maybe they can improve a little bit the solubility of your protein. If they not work you can really move to Baculo system or try another more soluble tag like NUS.

yang said:

Hello, everyone, I am trying to express a GST taged human protein domain in E. coli. The fragment is cloned into pGEX6p-1 and has correct sequence and reading frame. I have tried many ways including changes of temperature, IPTG concentration and even the host cells ( I tried BL21, BL21(DE3)-RP, BL21(DE3)-RIL). Finding the BL21(DE3)-RP worked out. But most of the objected protein is expressed in inclusion body,the soluble is a little.So I lower the temperature to 27℃.I only get 3mg objected soluble protein from 5 liter culture medium.Could anyone please give me some idea and suggestions how to improve the soluble protein expression? Thanks a lot.