His-rich E.coli Proteins

Eric C. Anderson anderson at pharmdec.wustl.edu
Sat May 20 14:57:49 EST 1995


hi there,

i've been reading a little here about histidine rich E.coli proteins
co-purifying or outcompeting recombinant 6XHis-Tagged proteins expressed
in E.coli when run over a Ni2+-Agarose column.  what i'm wondering is if
anyone has the apparent molecular weights of these proteins, especially
ones that have come from Top10 F' E. coli.

my problem is as follows.  i have a His-Tagged protein expressed in Top10
cells using Invitrogen's pTrcHisC vector.  when i induce the cells and
then pass the lysate (obtained using glass beads in the presence of
Pefabloc as the protease inhibitor) over Qiagen's Ni-NTA spin columns and
then elute in 25mM, 50mM, 100mM, 150mM and 250mM Imidazole, i get what
appears to be my protein of interest (and has activity in an HPLC assay)
but in the 25mM-100mM fractions, i get 2 significan bands at about 45kDa
and 50kDa.  anybody else seen these?  any suggestions on how to get rid of
them?

any ideas on what this is or what can be done about them will be greatly
appreciated.

thanks in advance for any help.

eric

-- 
"i don't know what caffeine does for you, but i'm pretty sure that without it your head caves in."

eric c. anderson                                 anderson at pharmdec.wustl.edu
dept. of molecular bio. and pharm.               (314)362-3963 (lab)
washington univ. school of medicine              (314)362-7058 (FAX)
660 s. euclid box 8103                           
st. louis, mo 63110

for a comprehensive list of bike related web pages... http://pharmdec.wustl.edu/~anderson/anderson.html



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