Can't express protein

Steven Goldberg goldberg at bms.com
Thu Nov 9 10:40:46 EST 1995


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 





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