Problem expressing a 50-60 kDa mammalian protein in E. coli... tips?

[Graham Dellaire, Ph.D.]

Dear all,

I am trying to express a 1.65 kb cDNA in E. coli using the pET32+ system
with BL21(DE3) bugs.  The protein including the trxa/his tag should be
about 50 to 60 kDa.  The protocol I used is as follows: first I grow up
a 5 ml culture overnight from a single colony; then in the morning to
inoculate 10 mls of medium with 100 ul of the o/n culture; two hours
later I start induction with 1 mM IPTG and collect samples at 1 h, 2 hr,
4 hrs post-induction and o/n.  By coomassie staining I see basically no
induction at all.  

The protein sequence has quite a few arginines and lysines (so fairly
basic) and perhaps this is a problem, although solubility may not be an
issue!

If anyone has any experience in expressing a large protein in E. coli I
would like to hear how you got the thing to be expressed efficiently.  I
am currently trying 0.1 mM IPTG induction overnight at 37 and also I am
trying the host strain ad494(DE3) and ad494(DE3)-lys.

Any and all tips/protocols are much appreciated.

Cheers,

Graham Dellaire
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Graham Dellaire, Ph.D.

MRC Human Genetics Unit
Western General Hospital
Crewe Road
Edinburgh, UK
EH4 2XU

Fax: +44 (0)131 343 2620   
Phone: +44 (0)131 332 2471
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