Hallo all,
i've tried to express an enzyme (heterodimer, 150 kDa together, soluble, intracellular) using the cellfree/in-vitro Systems from NEB, PUREexpress-System (E.coli-like) and from Qiagen, EasyXpress-System (Insect-like). As the enzyme is higly active with a high turnover number the dimerization and the proper folding should be determined by a radioactive enzyme assay. But until now I can only confirm the expression of both subunits of the heterodimer by western-blot. The blots look fine as there are only one spot for each subunit and the molecular mass is correct. As the expression of the enzyme failed using BL21-Cells (E.coli) I wasn't surprised, that the NEB-System also failed, but why the Qiagen-System shows no hint of enzyme activity is not clear for me, because the Sf9-Insect-cells mostly used to express the enzyme works always fine. I've spoke with the technical service of Qiagen, if they could give me some information about the chaperons in the system, but they couldn't give me a definite statement about this. So, my question is, if somebody of you has tried a similar expression experiment and could give me some advice for my problem. Best regards, Jan _______________________________________________ Methods mailing list Methods@net.bio.net http://www.bio.net/biomail/listinfo/methods
