bacterial expression/protein purification

ChenHA hzhen at freeuk.com
Thu Apr 6 18:55:03 EST 2000


Benny Lo wrote:
> 
>
> How would one tell if the protein expressed is going into inclusion bodies?
>

If your protein is in the pellet after cell lysis and
centrifugation (if that is your question).  If what you want
to know beforehand if your protein is likely to go into
inclusion body, this is more difficult.  The presence of
disulphide bonds is likely to result in protein going into
inclusion body.  The presence of hydrophobic patches may
also affect solubility, but that is difficult to tell
without knowing the structure of the protein, although
structure prediction can sometimes help. 
 
> A separate question: I would like to express a recombinant protein in
> E.coli, ideally getting high yields in the supernatant (I can add a signal
> peptide in front of my protein). Is anyone aware of any good
> protocols/vectors for this job?

Generally, the presence of a fusion partner helps with
protein solubility, although lots of proteins are soluble
without any fusion partner.  If you are worried about
solubility, maltose-binding protein (use pMal vectors from
NEB) is supposed to be best at promoting solubility.  See

Kapust RB, Waugh DS, Protein Sci 1999 Aug;8(8):1668-74

Expressing your protein at low temperature can also help
with solubility.  There is, however, no guarantee that any
protein will be soluble.  You can always try to refold your
protein should it ends up in the inclusion body.




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