Hello.
I am currently trying to express a recombinant gene with Avitag at the N-terminal.
I'm aiming at a final product that looks like this: Avitag-(target protein)
The protein I use is already being expressed regularly in my laboratory. The protein is an IDP (intrinsically disordered protein).
I took the plasmid from the bugs, edited it so it has Avitag after RBS site, and confirmed colony formation after transformation. The plasmid was sent for sequencing and I confirmed the correct insertion of Avitag sequence in the correct place.
I followed the same protein expression/purification protocol for unmodified protein, and I got the recombinant protein to express (or at least I think it did). SDS-PAGE showed almost the same sized (not distinguishable) bands as expected since Avitag sequence is so short.
In order to completely make sure my recombinant protein is in fact what I want, I did TWO Western Blots using biotin antibody and target protein antibody as primary antibodies.
The recombinant protein was biotinylated using BirA enzymatic reaction (company protocol used). Western Blot was done using unmodified protein, non-biotinylated recomb protein, biotinylated recomb protein (3 lanes with ladder).
Western Blots with target protein antibody showed 3 bands as expected while biotin antibody showed nothing (1 band should have lit up).
I concluded it is either:
1. biotin is not present - biotinylation failed (but it's apparently very specific and highly efficient)
2. avitag is not being expressed - skips avitag start codon and starts with the next one down?
3. being IDP, avitag sequence is forming a weird structure at the N-terminal making biotinylation impossible
I recently repeated biotinylation and still got nothing.
I'm really stuck and don't know what went wrong.
Any inputs and ideas will be appreciated.
Thank you in advance!
Hi Keunmin
In my experience, when tagging proteins at the N-terminal, its always a better idea to delete the first MET codon on the protein. The first MET codon should preferable be seen before the Tag sequence. This said, the enzymes do not really skip the first MET after the RBS, so even if you did not remove the native protein's MET there should not be the greatest issue.
1. Did you sequence your Avitag-target protein clones (miniprep and then sequence) after transformation, just to check for correct insertion and that there was no point mutation (I know this is rare with high efficiency transformation commercial bacteria)?
2. How large is the Avitag and your target protein? What % gel are you using?
3. Do you have a positive control for this biotinylation reaction? (just so that we know that biotin was there and the BirA was working)
4. If the protein is known to have disordered regions, there is a good change that tags are going to be tricky, did you use adaptors or flanks to distance the tag from the protein?
I hope you can resolve this issue. Good Luck
Aparajita
Hello Aparajita
I did not know it was common practice to delete MET codon on native protein. Thank you for the tip!
To answer some of your questions:
1. Yes the transformed bacteria colony were grown as a new batch and miniprepped for sequencing
2. Avitag is 15 amino acid long while my native protein is about 450. I used 12% gel for SDS-PAGE. I can see protein bands fine with SDS-PAGE, but WB with biotin antibody is not working.
3. This is something I do not have. I actually didn't expect this stage of my experiment to cause any problems. I ordered some biotinylated ladder and avitag'd protein producing plasmids to have positive control. It's taking really long especially due to corona... I will update you on this when I get some results.
4. I did not give any particular linker (is this the same as adaptors or flanks you mentioned?) between tag and native protein because it is already disordered. I wanted to keep the protein as little modified as possible. Do you think a long rigid linker might be helpful?
Thank you so much for your input!
Ken
Hello,
- Maybe the tag was removed in vivo. Is a protease site present ?
- Maybe the biotin antibody is out. Try streptavidin-HRP instead.
- Did you try to purify your tagged protein on streptavidin-column or equivalent ? Maybe part of the recombinant protein is biotinylated and will be purified.
Hope will be helpful
Claude
Hello Claude Alban
The expression was done using BL21 derived cell. Not sure what you mean by "is a protease site present" but I did not give any protease. I also don't suspect any protease would be there.
I did suspect maybe biotin antibody is not working but I didn't do any positive control. I didn't have anything that can be used as control (eg biotin ladder) and I didn't expect this to cause me problems. This is being shipped now. Trying streptavidin-HRP might be worth a try too. I will note that down. I'm currently waiting for anti-biotin-HRP to arrive. (I thought MAYBE all the washing process might have caused something to go wrong)
I purified the protein using the standard protocol used to purify natural protein used by my other labmates. Ion exchange followed by HIC. Maybe it's better to just go ahead and biotinylate the product as it is being expressed and purify using streptavidin-column. Will expressing BirA together with recombinant protein automatically biotinylate the recombinant protein?
Thanks for the input.
Ken
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