Dear Tiancen,
This is perhaps a more extreme example of what many of us have experienced
over and over - namely that for some proteins very small changes make a huge
difference in expression, solubility, activity or all three :)
Long and rambling reply follows - don't read if you are easily bored :)
In my experience this phenomenon tends to be particularly prominent for
domains where it is fairly common to have less than 1% sequence difference
be responsible for a transition between soluble & folded protein and
insoluble/aggregated garbage. Over time I have convinced myself that this
phenomenon is strongly correlated with folding; on several occasions I've
had situations similar to yours and the version of the protein that was
insoluble when expressed in E. coli was very nicely behaved in insect cells
or yeast (without any changes in sequence!) - my best guess that chaperones,
different ribosomes, intracellular conditions, or some other complex
features of the transcription/translation cascade are different enough
between expression hosts and in some hosts the 'insoluble' version of the
protein can be properly folded.
Perhaps an even more extreme example of very tiny changes affecting protein
folding can be the difference in stability of reduced versus oxidized
metalloproteins. Specifically I remember reading a cytochrome research paper
where the authors took advantage of the marked difference in stability of
the two forms and conducted very elegant nanoscale folding experiments using
photo-induced oxidation (or was it reduction?) as trigger for folding. This
allowed them to study folding on very short time scales. The cool science
aside, this is a good example of how a *single electron* can make or break
an entire protein.
In practical terms this means that in construct design and expression
experiments there's always a need to balance the factors that may (and do)
influence folding in specific host(s) of choice. Some systems seem to be
more forgiving - in my opinion eukaryotic expression hosts possess more
advanced 'assisted folding' systems and therefore tolerate sloppy construct
design a lot more than prokaryotic ones. In a pinch (and I mean really, when
nothing else works and there are no orthologs left to be tried) I've been
known to slice proteins with 5 or even 3-aa step - for E. coli work; whereas
we got away with a lot larger window in eukaryotic systems. This is a good
thing since most eukaryots are a lot less facile as expression hosts and
therefore require more effort per construct.
Artem
-----Original Message-----
From: CCP4 bulletin board [mailto:[log in to unmask]] On Behalf Of
Tiancen Hu
Sent: Wednesday, November 25, 2009 10:27 PM
To: [log in to unmask]
Subject: [ccp4bb] Truncation of first two residues "rescued" an insoluble
protein?
Dear all,
Sorry for the off-topic question, but I am really curious why two
terminal residues could make such huge difference.
I am working on a all helical domain starting from residue 1 to about
130. It expressed well with N-terminal His tag in both E.Coli and
insect cell, but stayed insoluble in both hosts. The fusion of N
terminal MBP tag could produce soluble form, but the target protein
precipitated immediately after cleavage of MBP. I tried several
constructs ending at different C-terminal residues, but none helped.
However, when I truncrate the N-terminal residues one by one, the
deletion of Met1 and Ala2 turned this protein into almost "completely"
soluble (30mg yield per liter E.coli cultue), and it behaved good
after cleavage of N-terminal His tag. In homologous structures, the
first helix starts from the third reidues.
So my question is what property of the protein might have been
affected by the first two residues, the surface hydrophobicity, the
folding process, or something else?And would this one-by-one
truncation of N-terminus be commonly helpful when working on insoluble
small proteins?
Thank you!
Best regards,
Tiancen
Postdoctoral Fellow
Novartis Intitutes for Biomedical Research
250 Mass Ave
Cambridge
MA 02139
U.S.
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