His-tag protein purification

[Rubini Kannagara]

Hi all
 I have got some problems, and wonder if there is anybody that can help
me out.


 I have cloned and expressed two plant proteins in E. coli with the size
of 70 kDa and 97 kDa respectively. The
 DNA-sequences encoding the two proteins were individually cloned and
their respective proteins
 expressed in the expression vector pET-24a. This vector provides an
optional histidine-tag. The DNA-fragments were
 inserted in frame with the histidine-tag. Subsequently I managed to
express them in inclusion bodies, (with the right
 size). When trying to purify the 70 kDa protein over a Ni-NTA column
under denaturing conditions, the protein was
 eluted during a low stringency wash procedure with 50 mM imidazol. In
the protocol for Qiaexpress system it is
 stated that proteins
 containing a histidine-tag will be eluted with higher concentrations of
imidazol such as 250 mM. This led me to believe
 that something might be wrong with my his-tag.

 I did not fully sequence my fragment but only the ligation junctions to
ensure correct reading frame. My question:  is it
 possible with only the sequence of the ligation junctions to determine
if the fragment sequence is in or out of frame
 with the histidine tag. Doesn’t one need to sequence the entire insert
including the ligation junctions to establish a
 frame shift mutation?.

 Coming back to the 97 kDa protein. I only succeeded once to express it.
It seems to be highly sensitive and is
 subjected to rapidly
 degradation. I am expressing it in the E.coli strain Bl21 that is
suppossdly deficient in lon and omp -proteases. The
 lysis of the cells is done in the presence of 1mM PMSF. I have tried
numerous growth conditions without success.
 Can anybody hand me some advice on this matter?