contaminant when overexpressing a GST tagged protein

From: SANCHEZ BARRENA, MARIA JOSE
Date: 22 March 2012 15:40

Dear all,

I am trying to express a eukatiotic protein (E. coli codon optimized sequence) with a GST tag at the N-terminus. I always get my overexpressed protein and a contaminant around 60kDa. This contaminant is not washed out of the column when washing glutathione beads with 1M NaCl-buffer. However, during o/n incubation with proteases that cleave the GST off (thrombin or TEV), the contaminant is in the soluble fraction.....

Has someone had this experience? I know about contaminants that bind to Ni2+ when overexpressing a His-tagged protein, but this is the first time I get such thing with a GST-tagged protein.

One could think that that "contaminant" could be a protein that binds to my overexpressed protein, but I do not think so, cause I always get a huge band of the contaminant, independently on the amount of the protein of interest....

Many thanks in advance for all your suggestions and sorry for asking about non-crystallographic topics.

Regards,

Maria

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From: Antony Oliver

I would hazard a guess of Gro-EL.

With regards,

Tony.

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Dr Antony W Oliver

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From: Parthasarathy Sampathkumar


Hi Maria,

As mentioned by Tony, it could be a chaperonin. Having little of ATP (0.5mM or less) and Mg2+ (1mM) in lysis buffer might help.

Good Luck,

Partha