How to avoid inclusion bodies ?
zinc at zifi.genetics.utah.edu
Tue May 23 09:56:45 EST 1995
In article <3pqbq5$lu1 at n.ruf.uni-freiburg.de>,
Clemens Vonrhein <vonrhein at bio5.chemie.uni-freiburg.de> wrote:
>The subject says it all: I want to avoid the expression of a protein
>as inclusion bodies.
>As far as I read the literature, there are some rules of thumb:
>- lower temperature (30, 25, 20, 15 degrees Celsius ?)
>- low IPTG concentration
>- rich medium ( terific broth, ...)
>- high OD and short incubation times (OD=1.0-2.0 and t=1-2h)
>- adding needed cofactors (FAD, FMN, ...)
>Are these rules true in general ? Is there a good and recent review ?
>What are your own experiences ?
>Is it worth trying to get soluble protein ? Or is it better to get a
>lot of protein as inclusion bodies and try to refold it ?
>BTW, I use a pET-22/BL21 system.
>Any hints/answers/tips .. highly apreciated.
well, i work on a zinc finger protein (18 cysteines) and nearly all
protein is found in inclusion bodies under all conditions i've tried.
this also seems dependent on the presence of a proline rich region to
some extent since about half the protein is soluble without that
my personal opinion is that inclusion bodies are 'a good thing (tm)'
since they represent such a highly enriched source of protein. of
course they are worthless if you cannot refold your protein but i
think you ought to try that before writing off using inclusion bodies
as a source of protein.
i can refold by simple dilution or by dialysis against the buffer i'd
like the protein to be in. it's convenient that way i suppose.
anyway, i'd give refolding a shot first, just include any cofactors or
DTT if you have cysteines around. if the cysteines are supposed to be
disulfide bonded you'll probably have a problem getting the right
bonds formed if there are more than two :( .
patrick finerty -- finerty at msscc.med.utah.edu
pfinerty at nyx10.cs.du.edu
U of Utah biochem grad student/slave in the Bass lab
easily found at (801) 585-3110 almost anytime. rm 207 wintrobe.
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