In article <52690.M.Holland at dwe.csiro.au>, M.Holland at dwe.csiro.au ("Michael Holland") writes:
> We are trying to express sequence from a rabbit sperm protein in XL1-blue.
> We have no idea of the function of this protein and data base searching
> shows no significant homology with existing proteins. This isn't too
> surprising as it seems to be sperm specific and not present in other
> tissues. Plasmid minipreps carried out on pFLAG containing the sequence in
> frame give very low yields. When cells containing these plasmids were grown
> with IPTG the growth rate is slow. All this suggests toxicity. Where do we
> go from here? Can someone point me to a discussion of the alternative for
> dealing with this sort of problem?
> Michael K Holland PhD
> Principal Research Scientist
> CRC for Biological Control of
> Vertebrate Pests
> CSIRO, Wildlife & Ecology
> PO Box 84, Lyneham ACT 2602, Australia
> Fax: 61-6-242-9242 Phone:61-6-242-1793
>M.Holland at artemis.cbr.dwe.csiro.au
Take a look at the following articles:
FW Studier, et al., "Use of T7 RNA Polymerase to Direct Expression of Cloned
Genes," Methods in Enzymology 185: 60 (1990)
FW Studier, "Use of Bacteriophage T7 Lysozyme to Improve an Inducible T7
Expression System," J Mol Biol 219: 37 (1991)
The strategy for dealing with toxicity is to reduce the basal expression of the
cloned gene by inhibiting T7 RNA polymerase with T7 lysozyme. The expression
system described in these articles is available from Novagen, Inc., 597 Science
Drive, Madison, Wisconsin 53711, USA, telephone 800-526-7319 or 608-238-6110.
Gregg Wells
Department of Pathology
University of Pennsylvania
Philadelphia, PA 19104-4283
USA
email: pathology at a1.mscf.upenn.edu
