Amplified product of Coat protein of Cucumber green mottle mosaic virus was put in expression vector pET and transformation was done with Rosetta strain. Expression of coat protein is not occurring even after induction with IPTG when used at 1 mM concentration after 3 hrs/overnight induction. May I know the reason?
Hi,
Firstly how are you assessing expression (or not) is it a gel of total protein or is it a soluble fraction? If you are looking at total protein then you may have expression but at too low levels to be obvious on the gel, if you are looking at a soluble fraction then it could be in the insoluble fraction.
Do you have glucose 1% throughout (in plates and media) to fully repress expression until induced?-this can have an all-or-nothing effect on expression of toxic proteins
Do you have Rosetta or Rosetta (DE3)? Without the DE3 pET vectors do not work as there will be no T7 polymerase in your E.coli.
Transformed cells were put in 5 ml of LB and grown overnight. Later, 500 ul of overnight grown culture was used to inoculate 50 ml of LB for 4 hrs. 15 ml of LB culture was taken out in a separate falcon tube and allowed to grow. 35 ml of the LB culture left out in the conical flask was induced with IPTG @ 1mM concentration. 15 ml of Aliquot was taken after 3 hrs and bacteria was pelleted and dissolved in 1-2 ml of 1X PBS buffer. Rest of the 15 ml was kept for overnight growth which was also pelleted and dissolved in 1-2 ml of 1x PBS buffer. These aliquots dissolved in PBS buffer was subjected to sonication. Sonicated samples were centrifuged and both supernatant (soluble) and precipitate( insoluble fraction) were used for checking on the SDS PAGE . I didnt get corresponding band of 17 KDa. for the coat protein expressed. I will check back and get full details of the Rosetta strain since we borrowed the Rosetta strain from other lab.
The Rosetta strain could still be the problem if it turns out to not have DE3 integrated.
I assume also that the plasmid is fully sequenced and everything is in frame with the initiation codon or tag from the pET vector and has the correct stop codon.
You already have a nice comparison on the gels of induced/non-induced but if your expression levels are low (
Will definitely try and let you know the result after following your suggestions. Thanks for your time though
Best Regards
The problem is that I cloned the coat protein in pET 28A between the EcoR1 and Bam H1 site which is 17.3 KDa. However there are some codons of the Vector pET28A coming before and after the cloned gene dueto which the coat protein is not coming as single protein but its coming along with the vector sequence which is around 6.3 KDa in addition to 17.3 KDa. So the fusion protein comes as 23.5 + KDa instead of 17.3 KDa. I was searching at 17 KDa location in the gel which is not appropriate. I find out the mistake and accordingly i confirmed the protein expressing thru western blotting also. Thanks everyone for your suggestions. I also changed to pET 29A (+) vector and also BL-21 codon plus RIPL strain since my coat protein has rare codons which will be translated by this improved strain rather than BL-21 strain.
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