I am working with WD40 genes in cyanobacteria. I have cloned a WD40 gene of size 800bp in the pET28a vector and expressed it in BL21 (DE3) cells. I have induced it with 0.5 mM of IPTG. The induction has occurred as observed in SDS PAGE but the size of the induced band is lower than expected i.e. it should give band of size 33 kDa but instead it is showing 20 kDa. Any suggestions on how can I improve my protein induction.
You may recheck the construct you have used to confirm integrity of the gene introduced. This can be easily confirmed by Sanger Sequencing of the construct. Hope this solves your problem with protein expression.
Dear Krishna,
few suggestions:
1) as Akhil advised, check your construct;
2) do the Western Blot to confirm that you really have expression of your protein, because IPTG changes expression pattern of bacterial proteins as well;
3) your protein may get proteolised, so checking the band with mass-spec would both tell you if its your protein, and if it is - where is has been digested;
Sometimes it happens, that SDS-PAGE doesn't show the correct mass of the protein. The famous example is p53, which has a mass of 43,7 kDa, but it migrates at 53 kDa. So there is slight hope, that there is no problem :)
Good luck!
Dear Krishna
to give reliable suggestions we need to know more details about your protein sequence, induction condition and see at least the protein gel with eyes.
However if your clone sequence is it correct is possible that:
1) you have an issue of degradation by proteases (this happen generally if the protein contain some non structured regions that are more susceptible to proteases degradation). In this case one simple trial (if you are performing 37°C induction) is try to repeat the expression at lower temperature (eg. 25 or 17°C) or lower induction time and see if the high MW bands come out
2)You have a problem of rare codon (you can easly check it using a rare codon prediction tool avaialbe on line) inside your protein and your translation is interrupted. In this case you can try to use a strain able to provide rare codon as Codon+ or Rosetta E.coli
good luck
Manuele
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