I am assuming that you have already checked for all sorts of 'simple'
problems, such as spontaneous mutations in the promotor region, frameshifts,
etc. Could it be a nasty case of proteolysis ?
A.G.E.
<danielak at intra.niddk.nih.gov> wrote in message
news:20020910191745.1216.qmail at ww02.hostica.com...
> You can read in pET system manual that secondary structure of mRNA
transcript can interfere with ATG translation codon or RBS and can lower
expression level. I have trouble to express app. 14 kDa bacterial protein,
which supposed to be not toxic (al least its homologs from different
bacteria are not) - I am using pET28a vector and do not want to add any
tag - so I was looking for a reason for not getting any protein (I tried
different conditions with temperature or IPTG concentration or cells). Codon
usage looks not so bad, but when I did estimation of secondary structure of
mRNA using mfold program (found on Prof. Zuker's web page), I found always
some sequences complementary to ATG or RBS. Also with mfold found on another
web page I've got different results. I've already tried to "improve" the
secondary structure of mRNA making some silent mutations in regions, which
were complementary to ATG or RBS, but it did not improve protein expression.
I am still getting nothing. What do you think about it?
>>>>http://biowww.net/mynews/tree.php?group_name=bionet_molbio_proteins&begin=0
