Internal RBS.

eb86336 at eb86336 at
Mon Sep 12 19:19:12 EST 1994


Have you tried playing with copy number?  pBR322 is only a low copy plasmid,
may get more protein in higher copy plasmid.  

Where is this alternative RBS in the gene?  Is there an ATG 9+/- 1 nt from this
putative SD sequence?  If not then this putative RBS is probably NOT a problem. 
You could be having a problem with trying to express a gene that uses poorly
used E. coli codons, as you have stated that your gene is high %AT. 
Alternatively, there could be mRNA secondary structure that covers the RBS of
your gene, thereby severely inhibiting proper translation initiation.  To get
around this second issue, you could use a 2-cistron, translationally coupled
gene approach.
I would need a bit more information about your gene before I could come up with
other possible explanations for your problem, or tell you if the alternative
RBS scenario sounds plausible.

Mike Menke
Eli Lilly and Company

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