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pMal-c2, modification of

ChenHA hzhen at freeuk.com
Sun Jan 30 16:03:12 EST 2000


Wolfgang Schechinger wrote:

> So it's probably a good idea to keep the context before the
> translation start the same as it is in original vector.

Umm, yes, but that's not what I said.  To make things clearer,  I would
make the following points:

There are many aspects of protein expression in e.coli, e.g.
transcription, translation an post-translational events such as folding,
secretion, protein degradation etc.  The use of N-terminus fusion partner
remove the need to worry about one aspect of protein expression, namely
translation initiation, as the fusion partner is well expressed and
therefore translation initiation is not an issue.  Fusion partners also
have the other advantages I mentioned such as increased solubility and
stability etc. and of course, provide useful methods for protein
purification.

However, I personally prefer not to use a fusion partner.  But expression
without a fusion partner is more problematic in that there are more things
to worry about, and it is often a matter of luck whether you get highly
expressed soluble protein.  For optimal translation initiation, you can
try, for example, making conservative mutations by replacing G/C for A/T
at the first few codons wherever possible, or select the best codons (such
as GCT and AAA at the second codon position) for protein expression.  This
can be conveniently done by designing oligos carefully when you do PCR
cloning.  There are other possibilities but they may be much more work or
require the use of other vectors.  But there is no reason why you could
not try expressing your protein directly without any modification because
many proteins have been successfully expressed in this way, and making
modifications to your DNA sequence may be a matter of trial and error with
no guarantee of success.







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