Expression of multimeric proteins

Per Mygind perm at biobase.dk
Mon Apr 21 12:31:58 EST 1997


Dear Netters

For a long time, I've enjyed the wealth of knowledge of this newsgroup,
and know I've got a problem for you all.

I have been working with a fusion-protein construct (in the pET vector
system for expression) for quite some time now. The construct encodes a
protein from a 500 basepair insert, followed by a 6-mer histidine tag
for nickel-affinity purification. The expression in E.coli is great, but
in order to increase the size of the protein (for use as a diagnostic
tool in ELISA) , I want to express a multimer fusionprotein. At first I
cloned in another 500 basepair fragment (identical to the first insert).
It resulted in very little expression, but still detectable on a
coomassie-stained SDS-gel. I happily continued, making my construct a
trimer of identical protein-sequences. To my surprise, it resulted in no
expression, whatsoever. 

In short terms

monomer: 1 X (15kDaProtein)-Histag	Good expression

dimer:	 2 X (15kDaProtein)-Histag	Little expression				 

trimer	 3 X (15kDaProtein)-Histag	No detextable expression

WHAT IS GOING ON ???????????

Surely the monomer is not toxic (thats what the recombinant coli teels
me), hardly any degration products are seen. The clones have been
sequenced thoroughly from both ends, and the sequence seems to be okay.
(so its got nothing to do with recombination) Is there anything in the
expression itself, that would permit expression of such multimers ? I'm
thinking in terms of DNA/RNA structures ? 

Anyone out there, please make comments on this and post it to me

With Regards

Per Mygind, Cand.scient.
Department of Medical Microbiologi and Immunologi
University of Aarhus, Denmark

e-mail: perm at biobase.dk



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