In article <47t7du$1lt at synapse.bms.com>, 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?
>> Any help would be greatly appreciated.
>> Steve Goldberg
An essential first few steps to check expression ( many or all you may
have tried already):
-Make sure strains are correct, (ie DE3 background for pET, mutant lambda
repressor for Pl)
-Make sure you have correct plasmid, to begin with AND at induction when
antibiotic is long gone.
-Make sure you have sequence, in frame, unmutated.
-Make sure you have mRNA: probe northern with end labeled PCR primer.
-Look for product with antibody (to leader peptide?) or a pulse chase to
see if your protein is made in tiny amounts, quickly degraded, or both.
I see no obvious problem with the first few codons and codon bias is
usually a nonproblem in E.coli unless you're talking multiple Arg
residues. If your mRNA is incredibly unstable you may bennefit from the
retro regulator element from B. thurinigensis (sp?). It is also possible
your product is incredibly toxic, the original Studier ref. on pET
addresses how to work with this. Good luck.
PS few human proteins respond well to the the higher temps used with the
Pl promoter system, temps <37°C are often the ticket for good expression.
Institute of Molecular Biology
University of Oregon
tedm at darkwing.uoregon.edu