Hi all,
I expressed a protein of molecular weight 61 kDa using pET28 plasmid (kanamycin resistant) using XbaI and BamHI restriction enzymes in E. coli BL21 (DE3) at induction conditions- 0.2 mM IPTG, 25 degrees C, and 120 rpm (checked using SDS-PAGE). But when I am expressing the same protein using pET43 plasmid (ampicillin resistant) using the same restriction enzymes in E. coli BL21 (DE3), I am not getting any expression or bands on SDS-PAGE.
The gene is sequenced and there is no sign of mutation or orientation issue.
Both the plasmids have the same T7 promoter, the only difference is the antibiotic-resistant marker.
Why am I not getting any expression in E. coli BL21 using pET43 but getting abundant protein using pET28?
Assuming that your constructs really are correct, I would suggest you retransform the pET43 clone and pick a few independent colonies and test those. Sometimes a transformant just stops expressing or fails to.
I agree with Michael J. Benedik: it's probably best to reclone and pick a few independent colonies.
Even though your gene of interest might not have any mutations and is in the correct orientation, did you confirm that it's in-frame with the T7 promoter? If it's not in-frame, your protein won't express.
Also, having a quick look at the pET-43.1a(+) vector map (compared to pET28), I see that the XbaI and BamHI cut-sites are quite far apart, and if you used those two to cut your pET-43 vector for ligation, it looks like you might have cut out a "ribosome binding site" at positions 2326-2331 at the same time. Maybe that's part of the issue.
The positions of BamHI and XbaI in the pET28 vector are much closer than their positions in the pET43 vector - you seem to be cutting out quite a huge chunk from the pET43 vector if you keep those cut-sites.
I would try Michael's suggestion first, and if you still don't get expression, consider using cut-sites in pET43 that are closer together.
Dear Ankita Mi
just some comments and questions to undestand better:
Are you sure that the only differences is the antibiotic resistance and there are not difference in the sequence of expressed contruct (eg tag?)
Did you produced C_terminal tagged (eg His) or untagged proteins?
i'm not expert of pet43 but from a fast look to the vector map:
- First of all i do not undesrtand exacltly as also with pet28 you can obtain a good expression since if i'm not wrong the digestion with Xba will remove the RBS site. Did You add it again in you forward primer?
How long is your induction and how much antibiotic did you add into the media?
Since kanamicin is more stable than ampuiclllin and some differences n could be observed with long inductions but it is strange that this happen at 20°C.
Unfortunatelly i do not have a ready solution but i hope that those comment may suggest to you some good ones.
good luck
Manuele
Thank you so much for your replies!
Michael J. Benedik Sure, I will re-transform the pET clone in BL21 again.
Dewald Schoeman I need to remove Nus-tag that's why I digested the plasmid with XbaI and BamHI. I have added the RBS site in my forward primer. Earlier I expressed another gene in BL21DE3 using pET43 digested with XbaI and BamHI and it expressed beautifully! As you and Michael suggested, I will re-transform it and check.
Manuele Martinelli I have added C-terminal His-tag. I have added RBS site in my forward primer.
Induction condition: 0.2 mM IPTG, 25 degrees C, 120 rpm for 7h
Antibiotic added: Ampicillin 100 ug/mL
Just want to update you all.
As per Michael J. Benedik and Dewald Schoeman 's suggestion, I retransformed the clone into E. coli BL21 (DE3) and got the expression. I did SDS-PAGE and got beautiful bands. I checked the activity of that enzyme and it is working perfectly. Seriously, sometimes a transformant won't express!!!
Thank you Michael J. Benedik , Dewald Schoeman and Manuele Martinelli for your suggestions.
Good to hear that it worked out and was so simple. I don't know the reason why (never really chased it down) but I have seen this phenomenon or lack of expression in some transformants quite frequently.
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