protein solubility - (Feb/21/2005 )

Hello,

I have a problem about protein solubility (inclusion body). The information of gene construct is as follows;

i) pQE-80L system (N-terminal His-tag)
ii) gene size, 1.5 kb (signal peptide removed)
iii) fully sequenced (no error)
iv) over-expressed by adding IPTG (0.001-1 mM)
v) 18, 25, and 37 degree incubation tested
vi) host cell, BL21 (DE3)

BUT all over-expressed protein is localized in insoluble fraction by all trials I couldn't detect enzyme activity from soluble fraction. To be strange, when I inoculated the cell on LB plate containing fluorescent substrate, it definitely showed fluorescence around the cell.

how can I figure it out? what's the problem?

Denature the inclusion body with 8M guanidium HCl or urea. dialyse of the compound and check for activity.

expression might be too much. change the host cell.

Good Luck

Sharath.B
Research Fellow
National Chemical Laboratory
India