protein production and purification

Chen Ho An chen at bsm.bioc.ucl.ac.uk
Thu Oct 1 15:43:14 EST 1998


Cornelius Krasel (krasel at wpxx02.toxi.uni-wuerzburg.de) wrote:
: 
: As far as I know, this is only the extracellular domain of the TCR. It
: is true that E. coli can successfully express a lot of soluble proteins
: or protein domains, but has tremendous troubles expressing membrane
: proteins (with a few notable exceptions, for example bacteriorhodopsin
: or lac permease).

It is indeed the soluble portion.  However, that doesn't alter the fact
that many (yes, many) membrane proteins have been successfully expressed
in coli.  The latest one I saw is Beswick et al, Prot. Expression and
Purification, 13, 423-432.  Of course membrane proteins have always been
difficult to deal with, which is why there are so few structures of
membrane-bound proteins.   However, the advantage of coli over other
systems is such that many people are still trying to express membrane
proteins in coli even with the inherent difficulties.  

It also very much depends on what the original poster intended to do with
his protein, which is why I mentioned the need to clarify this point.
Which system to choose depends on how much protein he needs, what kinds of
studies it is going to be used for, what forms of protein he want to
expressed, cost consideration, etc.  

There is now a huge literature on expression in e.coli, it is therefore
important to know the possibilities that exist with coli rather than jump
straight into something that might cost more, take longer or makes less
proteins.  I don't, of course, claim that coli will work every time and
I've seen the failures and it is therefore important to know the
limitation of coli.  But then I've also seen other people makes
proteins in other systems (which takes a few years) and then find
that they can express the exact same correctly folded protein in coli
(which take a few months work).   Aaaaargh is the only word for it.

[mailed and posted]




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