GST fusion protein expression

Hello, everyone,
I am trying to express a GST taged mammalian protein domain (NACHT domain) in E. coli. The fragment is cloned into pGEX4T-2 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, Rossetta TM2, Rosetta gami 2). But none of them worked out. Could anyone please give me some idea and suggestions?
Thanks a lot.

Ping

Ping,

When you say "none of them worked out" does that mean you never see any protein by GST antibody either in soluble or insoluble fractions in your cells? How are you looking for the protein expression?

Have you looked to see whether you can make RNA off your construct via an in vitro transcrpition kit?

you might dig around this database for more anwsers also

http://www.embl.de/ExternalInfo/protein_unit/draft_frames/frame_flowchart_ext.htm

Let me know
RB

Hi, R Bishop,
Thank you so much for your suggestions and website. I did not use GST antibody to check the expression of GST fusion protein. Since I need high level of expression, I selected the coomasie staining. I am going to try the other vector pET41a to see if it can make some difference.
Thank you again.

I am working in Sinobio Biotech and expressed and purified more than 100 contract proteins. tagged or tag free. according to my experience, you may try other expression vectors. In most case, change to different fusion tag may resolve the question easyly since even you can detect the mRNA transcripted, you still couldn't get the protein you wanted.
fusion tag and the DNA sequence are most important for a foreign protein expression in e.coli.

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