fusion protein - expression problem!

[McNamara]

hey guys,

 

i got a fusion protein of a KR (25,5 kDa) and a FDH (44 kDa) linked by a polyglycine linker. in sds-page, there is not only the fusion-protein but also a strong band of the 44kDa protein.

do you have any ideas why that could be? maybe the linker degrades? but i didn´t find any proteases or stuff that cut polyglycine...

or is the template to long and polymerase falls off? but why is it always the 44kDa band that is seen clearly?

 

oh one more thing: i added a his-tag to the fusion protein and the recovery (estimated by sds-page) was only about the half! there was no overexpression at all...now how the hell could that happen?

 

you see, i have no idea=)

 

i´m thankful for any ideas! and if you have adequate papers - nice!

 

 

thanks,

McNamara

 

ps: sry for my english=)

[CharonY]

I need more info regarding type of fusion construct, how you overexpressed and how you purified it. I.e. do you mean that in the SDS page you only have the purified protein but you got two bands? Or is that the raw lysate.