Protein Expression Problem - N-end rule?? (Oct/30/2007 )
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 and then my protein. 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.
So you're saying there's no ATG codon at the start? That may be the problem; at the very least, it is unusual for a gene cloned into a regular expression vector, because they typically have the necessary transcription signals. What regulatory sequences does the vector have? For that matter, which vector are you currently using?
I am currently using the pMal-C2X vector which uses the strong tac promotor for expression of the fusion protein (MBP). There is the ATG start site after the phenylalanine.