ThioFusion Expression System

[Pieterke]

Who has experience with the ThioFusion Expression System for protein
purification, which is sold by Invitrogen? Is the fusion protein mainly
expressed in the soluble fraction? And is it possible to excrete the
proteins by osmotic shock treatment? And what is the best way to get rid
of the Thioredoxin in the protein mixture? Hopefully anyone can give me
some good advice. Thank you,

Pieterke

[Armin Sepp]

Dear Pieterke,
There does not seem to be any affinity tag in the ThioFusion vector as judged by their catalogue
data. I've also noticed people saying earlier in this newsgroup that the expression levels are, as
always, protein-specific.
If it is of interest, then Novagen sells analogous system codenamed pET32 that in my case helped
to push up the level of expression from ca 50microg/l in the case of GST-fusion to ca
500microg/l soluble protein. It is of interest that the optimal host in both cases was
BL21(DE3)pLysS (Novagen) although the recommended strain for thioredoxin fusion is AD494. The
fusion protein (containing three cysteins)was functionally active and could be purified from the
lysis mix to relative homegeneity by Ni-affinity chromatography.

In summary, there does not seem anything that can be taken for granted, Screening a panel of
vectors and hosts still seems the only option.

Good luck
(needless to say, I am just a customer for the named and impled companies....)
--
* Armin Sepp, PhD \ *
* Department of Immunology \ "Every little helps." (Tesco) *
* Babraham Institute \ *
* Cambridge CB2 4AT,U.K. \ Armin...@bbsrc.ac.uk *

[Armin Sepp]