Bernard Murray, PhD
spam at 127.0.0.1
Mon Nov 29 02:27:21 EST 1999
In article <81rsr9$32u$1 at nnrp1.deja.com>, SaoTo <daniboyton at my-deja.com> wrote:
> Can anyone give me the significance/use for the following components in
> a plasmid, they are downstream of the promoter, and upstream of the
> MCS- thioredoxin coding sequence, enterokinase recognition site, His6
> coding sequence.
Thioredoxin (I have never used this)
Fusion with this is supposed to increase the solubility of some
expressed proteins (to stop it all accumulating in bacterial
inclusion bodies). I believe that co-expression (rather than
a fusion) can be used instead.
*Theoretically* allows you to chop off any extra sequences
fused with your protein of interest (purification tag, affinity
tag etc. etc.). Actually, clean cleavage is not guaranteed
and is unlikely to be quantitative - but if you need quick
purification and also need to chop off the tag...
When your protein of interest contains tandem histidines it
can (usually) be purified quite easily using metal-affinity
chromatography (frequently using a "Nickel" column). The
usual tag contains 6 histidine fused to either the N- or
C-terminus but six is probably more than enough.
Bernard P. Murray, PhD
bpmurray at cgl . ucsf . edu
Department of Cellular & Molecular Pharmacology, UCSF
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