pET vector cloning of hydrophobic protein gene

Yasushi OKADA yokada at kinesin.kaibo1.m.u-tokyo.ac.jp
Thu Aug 3 09:36:07 EST 1995


In article <3vltmi$qn5 at warp.cris.com> mike.holloway at stjude.org (Mike Holloway) writes:

> I have to conclude that some protein is being made, despite what
> anyone says about the T7 promoter not being used without the
> polymerase being pumped out, and that its toxic.  This has been the
> previous experience in our lab with this protein using other
> bacterial expression vectors.  Apparently this isn't an isolated
> experience, since several companies have recently come out with this
> thioredoxin fusion protein scheme that's supposed to solve
> everything.

Do you consider that the protein is made even in the bugs that do not
have T7 pol gene?  If you can subclone your PCR product in bugs
without T7 pol, you can induce the expression of the protein by
the infection with phage that have T7 pol gene.  Such phage can be
obtained from Novagen or Invitrogen.

Hope this helps.


--
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* Yasushi Okada, MD.           | Email:yokada at kinesin.kaibo1.m.u-tokyo.ac.jp *
* Dept. Anatomy & Cell Biology | Tel:81-3-3812-2111 ext.3336                 *
* Univ. Tokyo  JAPAN           | Fax:81-3-5689-4856                          *
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