I'm trying to produce GST fusion proteins to use in pull down experiments. The genes are successfully cloned into pGEX-6P-1 plasmids and confirmed by sequencing and transformed into BL21 Star E.coli for expression. I cannot, however, seem to produce protein by induction with IPTG. I have tried a range of IPTG concentrations up to 2mM and induced at 28oC, which was recommended to me. When I Western Blot this using anti-GST antibody I do get bands, 1 at ~26 KDa which is roughly he size that would be expected for the GST on it's own (there shouldn't be any) which increases in intensity with time but not IPTG concentration. There is a strong band at ~50 KDa which I have no idea what it could be and sometimes I get quite a faint band above this of ~60KDa. The proteins I want to tag are between 54 and 60 KDa so it's probably not these.
I was hoping someone might have had similar problems or may have any general advise for me to produce the proteins that I want.
Hi Nicholas here i would like to suggest some point sthat might be helpfult o get expression of your protein:
1. Try at different temperatures (25, 30 and 37°C) instead of just only one. I was unable to get my protein induced at 18°C.
2. Look for the necessity of rare tRNA codons for the tranlsation of your protein. If this is the case, then the strain BL21 (DE3) RP or even BL21 (DE3) pLysS might be helpful for your protein induction.
3. Think of the necessity of chaperones for the expression of your protein. There are certain strains that can provide you the necessary chaperons depending the protein size to be expressed.
4. If nothing works, then might be the size of your protein is a problem itself. Its expression alongwith GST tag makes it a large sized and hence a problem to get it induced in the bacteria. If this is the case, then you might go for in vitro protein expression, Pichia (yeast) or the insect cells....you may try in th eorder given above depedning the facility you can get more easily and available to your nearby.
Good luck
What kind of protein are you trying to express? Membrane proteins are often pretty hard to get.
Hi Nicholas, as Muhammad said it seems to be that your protein has synthesis's problem. Perhaps, translation is stopping in the middle of your protein, as you did mention, maybe you have only the first 25 or 35 kDa of your fusion protein detecting 50 and 60 kDa bands, because your Ecoli has not enough tRNA that recognize the codon inducing codon bias trouble. In order to solve that, you could express your GST fusion protein on RIPL codon plus E coli from stratagene, which contains plasmids expressing tRNA that are rare in ecoli avoiding codon bias troubles (one of the plasmid need to keep in chloramphenicol in addition to ampicilin of your vector). Once, I used this strain to avoid problems of codon bias. However, you could still see by western blotting at meantime the band at 25kDa and 75kDa corresponding to GST and your GST fusion protein, respectively. 1mM of IPTG is enough to express GST fusion protein from pGEX vectors. Low temperature like 20 degrees are good in order to increase the solubility of your fusion protein (expressed slowly) if your protein is in unsoluble fraction (together to membrane's proteins)..Did you check that?...I hope this info can help you, Good Luck!
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