Protein Expression problem - N-End Rule?? (Nov/01/2007 )

Hi everyone,

I am trying to express an 80KDa Staphylococcal protein in E.coli but am having alot of trouble. I have tried attaching it to a his tag and a MBP but still cannot see expression. It has been sequenced to ensure it is in frame and RT-PCR has verified that it is being transcribed. I have also transformed it into a rosetta strain in case of any rare codons. I have altered the growth conditions many times including induction OD, length of induction, growth media, IPTG concentration etc. I have now read about the N-end rule and have realised that when I sub-cloned from a Topo vector into the expression vector, the first 2 codons trancribed are glutamic acid and then phenylalanine (from Topo) and then my ATG of my gene. I am aware that hydrophobic amino acids can cause proteolysis to occur. Could this be happening in this case? This lab is new to protein work so any help would be much appreciated.

Many thanks............

Some proteins are very difficult to express in bacteria.
But, a really good idea is to get a GST-protein (or whatever tag) from a colleague
that has already been used successfully and use that as a positive control.
If you can express that protein then you know your technique is fine.
if not then you are doing something incorrectly.
Positive controls may be a little mor ework but will save you enormous amounts of time in the long run.



Hi everyone,

I am trying to express an 80KDa Staphylococcal protein in E.coli but am having alot of trouble. I have tried attaching it to a his tag and a MBP but still cannot see expression. It has been sequenced to ensure it is in frame and RT-PCR has verified that it is being transcribed. I have also transformed it into a rosetta strain in case of any rare codons. I have altered the growth conditions many times including induction OD, length of induction, growth media, IPTG concentration etc. I have now read about the N-end rule and have realised that when I sub-cloned from a Topo vector into the expression vector, the first 2 codons trancribed are glutamic acid and then phenylalanine (from Topo) and then my ATG of my gene. I am aware that hydrophobic amino acids can cause proteolysis to occur. Could this be happening in this case? This lab is new to protein work so any help would be much appreciated.

Many thanks............
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