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Jab30  
#1 Posted : 21 July 2017 09:08:40(UTC)
GE'Jab30

Joined: 21/07/2017(UTC)
Posts: 1

Hi, I've been trying to express a gram positive PTS transporter in E. coli BL21 (DE3) using the vector pET15b (N-terminal His tag). There isn't any obvious band in the insoluble / soluble fractions of cell lysate and nothing is coming out of the HisTrap enrichment. I've tried altering the conditions of expression e.g. lowering IPTG conc, changing the antibiotic but to no ovail. It could be that the protein is toxic to the cell? Any suggestions on the best way to tackle it? Thanks a lot
GE Protein purification team  
#2 Posted : 24 July 2017 09:00:45(UTC)
GE'GE Protein purification team

Joined: 16/11/2015(UTC)
Posts: 99

Dear Jab30,
Your results suggest that your protein is not expressed or expressed at a very low level and not detectable. If you cannot detect (SDS-PAGE, Western blot) your his-tagged protein after HisTrap, this could mean that the His-tag may be not well exposed or hidden in the 3D structure of the protein.
Did you try to put the His-tag to the C-terminal end ? You could also use some detergents (0.2% Twin) to change the 3D structure in mild conditions. Experimental set-up for expression (IPTG conc, temperature, induction duration) or for purification could also be checked. Imidazole concentration in binding buffer could also be to high to allow a good binding of your protein to the matrix.
Sometimes, Ni2+ is not the best ion for purifying His-tagged and you can try Co2+ (HiTrap TALON) or other divalent ions on HiTrap IMAC. The first main thing would be to detect your protein to evaluate the level of its expression. If your expressed protein is toxic, it would interfere with the growth of bacteria.
Thank you.
Dominique Dutaud - Protein Purification Specialist, Scientific Support, GE Healthcare
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