1 reply to this topic

[lost_in_the_lab]

Hi!

I'm trying to produce a GST-tagged protein. Originally the protein in question had a C-term His-tag and I need to replace it by a N-term GST-tag. The original protein is very soluble and produces nicely. For some reason I can't get my GST fusion protein to go in solution. It is really bizarre since GST is supposed to promote solubility... I am currently testing my second construct (with a longer linker between the two proteins) and it still doesn't work... do you have any idea what I could do? I tried reducing incubation temperature during protein production and tried several lysis methods... and still nothing...


thank you for helping!

[pras45]

lost_in_the_lab, on Jan 28 2010, 06:36 AM, said:

Hi!

I'm trying to produce a GST-tagged protein. Originally the protein in question had a C-term His-tag and I need to replace it by a N-term GST-tag. The original protein is very soluble and produces nicely. For some reason I can't get my GST fusion protein to go in solution. It is really bizarre since GST is supposed to promote solubility... I am currently testing my second construct (with a longer linker between the two proteins) and it still doesn't work... do you have any idea what I could do? I tried reducing incubation temperature during protein production and tried several lysis methods... and still nothing...


thank you for helping!

at what stage of the purification does your protein start to precipitate?