Cell-free Protein expression

Amna Saeed asaeed at NEU.EDU
Tue Sep 22 18:56:55 EST 1998


Hello:
	I was wondering if anyone had successfully used a cell-free system for
protein expression? I am trying to express various "domain-swapped"
constructs of a soluble E.coli protein, but am not having much luck
assaying for activity. Recombinant protein yields don't look too good on
SDS-PAGE after 2hr IPTG induction (optimized for parental protein) and I
have tried PMSF and some other protease inhibitor cocktail from
Boehringer. I just need to express my constructs to see whether or not
they are functional but I don't know how I will do this if I can't even
get the protein out of the bacteria intact!!! I've tried sonication and
B-PER. The parental yields are always great. It's just that the
recombinants seem to be preferentially proteolysed or something. Would
greatly appreciate any helpful hints. Thanks a lot, Amna.



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