IUBio

baculo expressed protein insoluble

Dima Klenchin klenchin at facstaff.REMOVE_TO_REPLY.wisc.edu
Fri Mar 26 14:52:41 EST 1999


:Dear reader,
:
:I have a serious problem to express a set of His-tagged deletion mutants of
:my protein in the baculo-system. All fragments are insoluble as judged by
:comparison of native and denaturing protein purification methods. 

This is wrong criterium. If it is insoluble, then you should not see it in 
cell cytosolic fraction, but in pellet instead. The inability to bind at 
native consditions is frequent and is caused by intrinsic protein 
folding that results in poor accessibility of tag while the protein is
perfectly soluble. Try putting tag on C terminus instead of N

:Under
:denaturing conditions (6M urea)I see a huge expression of the proteins but
:there is no binding to the metal-affinity substrate under native conditions.
:Interestingly I can see little round "inclusion bodys" (?) in the infected
:insect cell culture (is that possible ?). 

Never heard of inclusion bodies in insect cells. Inclusion bodies 
are caused by aggregation of misfolded protein when present at high
concentrations. If a protein is eucariotic, this is rather unlikely. 
In any case, insect cells would rather degrade the protein rather than 
accumulate huge insoluble aggregates. 

;To tackle this problem, I did an
:infection time course to figure out whether a portion of the protein is
:soluble in early stages of infection. Unfortunately it was not. Since I use
:Invitrogens Hi5 insect cell line with is supposed to express 5 to 10 fold
:more protein I want to test a "normal" SF9/X/21 cell line. 

I belived this myth for a while too. After careful comparison, this is 
what I found: High5 express manyfold more protein than sf cells
ONLY when infection levels are low (faster virus propagation in them?).
With high multiplicities of infection, the difference basically disappears
resulting in protein-dependent preferences within only factor of two-fold.
(Sometimes SFs work better, sometimes High5)

:Also the decrease
:of culture temperature to room temp. might slow down the protein synthesis an
:lead to soluble protein. 

Prtactically matters only in procariots. Eucariots are picky about 
temperatures. High5 in my hands do not like temperatures below 25C
(almost stop dividing), while Sfs are only moderately slowed at down 
to 20C.

:Since I want to check a enzymatic activity,
:refolding or renaturing is not the method of choice. 

For some enzymes activity is restored to over 90%. Worth checking
because in this case you can get 100X more stuff much cheaper. 

Does anyone has
:experience with that kind of problem ? I appreciate any suggestions

Try working with lysates (say, in 1% non-ionic detergent; many enzymes
work in such detergents quite nicely). Also a possibility to crudely 
fractionate cytosols/lysates to the purity sufficient for enzymatic 
assay.

Good luck,

        - Dima




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