In article <goldberg-0305961550350001 at 140.176.74.203>, goldberg at bms.com
(Steven Goldberg) wrote:
> I have been trying (unsucessfully) to express a mammalian esterase in
> Pichia pastoris both as a fusion to the S. cerevisiae alpha-mating factor
> and as an intracellular protein. I have sequenced and everything looks
> fine. I have read that some eukaryotic proteins cannot be expressed due
> to premature termination at AT-rich regions within the gene of interest.
> My question is what is considered to be an "AT-rich" region (i.e., is it a
> certain number of consecutive As or Ts, or larger area of high AT content,
> or both). I do have a sequence "AAATTTTTAA" which has caught my attention
> as a possible problem area.
>> Any ideas or opinions??
>> Thanks.
>> Steve
The sequence you report may be a problem, but the quickest way to find out
is to do a Northern blot. If you have abundant, shorter-than-expected
message(s) then premature transcription termination is likely the
problem. A single, discreet RNA product could point you toward the
culprit sequence, which could be "corrected" by mutagenesis. Other fixes,
for example for many termini, are much more labor intensive. Check out
Scorer et al, Gene 136:111-119 (1993).
Rich Buckholz
