cellfree/in-vitro-expression of heterodimer

Jan R. Kraehling via methods%40net.bio.net (by JanKraehling from alice-dsl.net)
Sat May 15 08:42:59 EST 2010


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

 



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