Steven Goldberg <goldberg at bms.com> wrote:
>I have been trying to express a mammalian protein in E. coli using both
>the pET system and another plasmid with the lambda pL promoter. The
>protein has an 18-amino acid leader; it was modified by PCR for
>ligation into an NdeI site in each instance, so that the mature coding
>region immediately follows the ATG. This was verified by DNA sequencing,
>and no base changes or deletions were seen. Following recommended
>protocols, I see no protein at all being produced using SDS-PAGE and
>Coomassie Blue staining. Since these promoters are both very strong, I
>am now looking to see if something within my gene of interest is
>preventing transcription and/or translation. It may be of interest to
>note that the sequence following the ATG is GGG CAG CCA GCC...(Gly Gln
>Pro Ala). It there something in this sequence (such as the GGG directly
>after the initiation codon) which could kill transcription or
>translation? Should I modify this region by adding a few amino acids
>before the Gly or by changing the first few codons to include more As
A few suggestion...
i) Are you sure the plasmid hasn't selected against expression in some
way? You could try an E.coli (maxi or mini cells) or in-vitro (zubay)
assay to check?
ii) Can you expect the protein to be lethal? It may select out as soon
as you put the induction on.
iii) Try a fusion. There's load available but remember that cleavage
may be difficult or expensive.
iv) Are you sure the protein isn't just going into inclusion bodies?
Make some and have a look
v) Try something fancier like yeast, Pichia or Baculoviruses. It might
be quicker than doing a load of manipulation.