Hi,
I am working on a 60 kDa C. elegans protein that is predicted to be mostly
alpha-helix. It is over-expressed in E.coli and the yield is about 1 mg/L of
cell culture. The CD spec at 4 degree showed the presence of dominant
alpha-helix. However, we dont have any functional assay to confirm that it is
folded correctly.
It is over-expressed as a GST-fusion. We noticed that after cleavage of GST,
it will easily precipitate if moved to room temperature (solution turns
cloudy). Otherwise, it is OK at 4 degree. The CD temperature melting experiment
showed a gradual change of signal, no sharp transition was observed. We later
found out that including some detergent in the buffer will make it stay soluble
at room temperature and showed as a dimer on SEC (4C or RT). Glycerol at 10%
will help too but not as good as detergent.
My concerns are, first this protein might not folded correctly, second, the
presence of probably high concentration of detergent in the final sample will
harm crystallization since the detergent will be co-concentrated with the
protein. I am wondering whether anyone has deal with proteins like this before
and their experience on improvement of the biochemical behavior and of course
crystallization. Many thanks.
Best,
chen
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