Marked
differences between in-vivo and in-vitro expression are observed!
1. The RTS systm gives higher
protein yields than expression in Rosetta and Origami strains. This is
evident by Coommasie staining (though you may claim that the in-vitro samples
are more concentrated following initial purification on glutathione beads).
2. Proteins synthesized
in-vitro are soluble as opposed to proteins synthesized in-vivo, though
no special additives (chaperones, detergents etc.) were added.
3. Only shorter fragments
of protein were synthesized in-vitro, as opposed to small quantities of
full length products, which were observed in-vivo (especially in lower
temperatures). Truncated proteins are found following premature falling
of the ribosome. This phenomenon may occur when proteins of mammalian origin
are translated in a coupled transcription -translation bacterial machinery.
Folding of the proteins, mRNA structure and presence of rare codons may
account for this phenomenon. In order to address these problems, we can
reduce the reaction temperature; Add chaperones, chemical chaperones, glycerol;
Add tRNAs for rare codons (as was done in this experiment).
Q7
Based
on all these expression results and on the initial bioinformatics data,
how do you suggest to continue this expression project?
1.
Needless
to say - abandon it !!! I was right all along !
2.
Consider expressing it as a fusion protein or as a secreted protein in
the same or additional bacterial strains.
3.
Consider expressing it in insect cells. (How?)
4.
Improve expression in-vitro. (How?)
Please
submit your answers by 19.2.04.
1.
You may be right, but you won't get a grade for this tutorial if you don't
continue.
Return.
.