GST-tagged protein expression problem-plz help - (Nov/07/2008 )

I am making a GST tagged recombinant protein in E.coli. I cloned my gene in pGEX-2T vector and checked the sequence. Then I tried to express in E.coli using BL21 star competent cells. Using both CBB and western blot I could detect a band thats size is slightly higher than GST band. I used 37 degree/25 degreeC growth conditions but the same result. I tried IPTG 0.1 mM to 1 mM condition.

Then I used Rosetta strain to overcome rare codons problem if there is any. Actually there are some rare codons in my construct but even using rosetta I got the same result. I am sure the gene is in frame.

please give me some suggestions to solve this problem.

My gene is of mouse origin and previously in our lab ( 5 years before) a GST tagged protein of human origin was successfully made.

I am now thinking to change the tag to His but not sure of its success.

plz help.

regards,
hasina

you can try PET32 vector or pet43,maybe they can work

Dear Hasina,

I am little confused with ur question . But anyway i will answer the question in the way i understood.

See when you clone with GST Tag , it will express GST fusion protein along with your protien . So you have to consider the Molecular weight of GST(~25Kda) plus your protein molecular weight. so when you add the 2 protiens MWs obviously your protein will appear slightly at higher side in CBB or in Western. because the protein was tagged with GST and Western primary antibody will detect it and binds successfully. ( this will tell you that the expressed protien is yours)

Correct me if iam wrong

Awaiting for your reply,

Regards
Sudhakar M
sudhakar8@gmail.com

Thx for the replies.

To Sudhakar, I want say that I was expecting a band (including GST tag) of 60 kDa but I got about 28 kDa band that is slightly higher than GST band ( if I check only GST expression).