pET expression problems

Chen Ho An chen at bsm.bioc.ucl.ac.uk
Thu Sep 3 09:48:57 EST 1998


Adrian Clarke (adrian.clarke at plantphys.umu.se) wrote:
[snip]

: The problem I have is that the sizes are strange - around 25-26 kDa for
: all constructs when I run a SDS-PAGE gel, whereas I expected 33-34 kDa
: from
: the full length clones and 30-31 kDa from the others. 

Did you do a mass-spec to check that it is actually only 25-26 kDa?  There
are a couple of possibilities if it is - the protein may be degraded,
though that is unlikely if it is actually in the inclusion body.
Therefore the next question is - is the degraded protein refolded?  It 
may be degradation and also some proteins can undergo autolysis.  
There is also the phenomenon of premature termination of protein
translation due to the presence of rare codons such as AGG and AGA
if they are present in tandem on in clusters.  Check your gene sequence
and only the C-terminus should be missing if that is the case.  






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