Can't express protein

Chris Jones mbzcaj at unix.ccc.nottingham.ac.uk
Fri Nov 10 04:04:04 EST 1995


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 
>and Ts?

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.

Good Luck!

Chris Jones






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