I am expressing 6xHis tagged 8.5KDa human Protein (pI~4.19) which cloned into pET21b expression vector and express in BL21 DE3 Codon plus strain. I see expression when I mix bacterial pellet with SDS Gel loading dye(Gel loading buffer contains 0.05% bromophenol blue , 40% sucrose, 0.1M EDTA (pH 8.0) and 0.5% SDS) and heat at 95°C for 5 miutes and then load on 15%SDS PAGE. But when I lysed it with lysis buffer (500mM NaCl, 20mM Tris pH 8.0, 2mM Benzamidine and 10 µg/ml Soyabean Trypsin inhibitor) and then sonicate it with pulse 2 Second ON and 12 second OFF For 6 Minutes with 32% amplitude, I am not able to see expression in supernatant as well as in pellet.
I also purified it with Ni-NTA beads and elute with 150, 250, 350 and 450 mM Imidazole but nothing was detectable on gel.
I also monitored OD of Induced and Uninduced culture for see whether my protein is toxic to cell or not and Uninduced culture grow at double rate compare to Induced culture.
Please suggest me-
1. Is my protein toxic to bacterial cell?
2. Is there any relation with pI of protein and pH of lysis buffer? As my protein pI is 4.19 and I am using lysis buffer with pH 8. If yes then what will be suitable pH for lysis buffer and its composition?
3. Whether my protein is express or not?
All gel picture and OD data is attached with word file.
It looks like your protein was not expressed after induction. The difference in growth curves between induced and uninduced cells suggests that your protein is toxic to the cells. You might try inducing the cells at room temperature or below instead of at 37oC. The slower growth may allow some expression of the protein. You might also try expressing it as a fusion protein (with a cleavable linker). The fusion protein may be less toxic. Another approach is to express it with an export signal sequence so that it doesn't accumulate in the cytoplasm.
It looks like your protein was not expressed after induction. The difference in growth curves between induced and uninduced cells suggests that your protein is toxic to the cells. You might try inducing the cells at room temperature or below instead of at 37oC. The slower growth may allow some expression of the protein. You might also try expressing it as a fusion protein (with a cleavable linker). The fusion protein may be less toxic. Another approach is to express it with an export signal sequence so that it doesn't accumulate in the cytoplasm.
@Adam B Shapiro Sir I had already checked induction and OD at 25 degree and 16 degree. Gel image also attached with this for the same. please have a look on that and then please give me suggestion. I also tried purification of same protein with GST tagged but after tag removal I don't got my protein thats why i cloned it in pET21b Vector.
@Arbind Kumar Sir I had already checked induction with 1, .5 and .2 mM IPTG conc. and OD at 25 degree and 16 degree. Gel image also attached with this for the same. please have a look on that and then please give me suggestion. I also tried purification of same protein with GST tagged but after tag removal I don't got my protein thats why i cloned it in pET21b Vector
I guess You have some reasons to use the codon plus strain. Sometimes it is better to synthesize ORF which is optimal for E. coli. Secondly, it is very good to have a control construct, which efficiently produces a protein in this system.
Hi Mohd, how do you know that your 8.5 kDa protein is being expressed?
Are you evaluating the expression of your protein by looking for stained bands on SDS-PAGE that are the right MW? Or do you detect your expressed protein by Western blotting?
@Steingrimur Stefansson Sir I am evaluating the expression of my protein by looking stained bands on SDS-PAGE.
Hi Mohd, so you don't know whether the ~8,5 kDa protein that you see on the SDS-PAGE is your protein of interest?
If you have an antibody against your protein, I strongly suggest that you do a western blot to confirm that your protein is being expressed.
Chasing after bands on SDS-PAGE will get you nowhere if they are not the protein you are trying to express.
I agree with Steingrimur that you need to be sure the protein is being expressed. The band at about the right MW could be something else. If you don't have an antibody to the protein, you can buy one to the His-tag. Alternatively, if the protein has a measurable activity that can be assayed in a crude extract, you can test for it that way.
Since the protein does not accumulate as the incubation time increases, and the cells also stop growing, if this band is your protein its expression is toxic to the cells. Therefore, a good way to express it is to grow the culture up to high density before inducing, then collect the cells after 1 hour.
This human protein may be insoluble when expressed in E. coli. If this can't be fixed by expressing at a lower temperature, you may be able to dissolve it in detergent from the pellet. Alternatively, it may be soluble but prefer lower salt than you are using in your lysis buffer (500 mM). If it is absolutely necessary due to the insolubility of the protein, you may be able to denature it fully in 6M urea, purify it on the Ni column, and refold it. Since it is a small protein, this has a pretty good chance of working. Does the protein have disulfide bonds? If so, you will have to reform these correctly, an added complication.
Speaking of disulfide bonds, I notice that your lysis buffer has no reducing agent in it. You should probably add some to avoid forming unwanted disulfide bonds between proteins.
One last thing: How confident are you that your sonication procedure is effective? It seems pretty mild.
@Adam B Shapiro thank you sir for giving me a valuable suggestion
Dear Mohd,i have some suggestion,use control for expression,before induction a,nd without inducer,for better judgement.anyway i say examine 1mM IPTG,OD0.7 after8,12,18,21 hours and 37 degree for4,6 hours.another thing sonicate your bacteria for20 plus ON and 25 second interval for8 minute!you do it for only2 second ON!!use sonication buffer or lysisi buffer same as optimom pH of your protein.good luck
Hi Mohd,
You need to purify interesting protein in yeast. Insoluble proteins in E.coli can be soluble in yeast expression systems in many cases.
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