Dear Armando,
Two similar, but not identical experiences:
- we expressed a protein and got some ugly small crystals. Then the
Edman degradation results on the purified protein came in and turned
out to correspond to chloramphenicol transferase (same size as our
protein).
- expressed a protein and got very nice crystals, got a dataset with
cell parameters suspiciously like LyCl7 (lysozyme heptachloride, that
protein that crystallises as easily as NaCl). Solved the structure and
of course it WAS LyCl7. The LyCl7 was a minor fraction in the protein
prep., but still crystallised (also same size as our protein).
Fortunately, in both cases we only lost several weeks and this is only
a small percentage of our failures. Also, they are remediable ones,
most failed projects are because the protein does not express in
soluble form, aggregates aspecifically or simply refuses to
crystallise...
In summary, like others, I can only stress the importance of checking
by N-terminal sequence analysis, mass-spec, Western-blotting etc. that
"that band on the gel" is indeed the protein you want.
Greetings,
Mark
PS CIB in Madrid has a proteomics service we use. Becaus it belongs to
CSIC, they will charge you the internal tariff:
http://www.cib.csic.es/en/servicio.php?iddepartamento=27
Mark J. van Raaij
http://webspersoais.usc.es/mark.vanraaij/
researcherID: B-3678-2009
On 18 Feb 2010, at 18:49, Armando Albert de la Cruz wrote:
I am trying to overexpress a His-tagged protein of 29KDa in E.coli
(BL21-codon plus) and I end up with a highly expressed product that
of 43KDa that binds to the Ni-column. I also have nice crystals.
Does anyone have any experience on this.
Armando
