HIS TAG ISSUES

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is5298mih
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HIS TAG ISSUES

Post by is5298mih » Tue Mar 28, 2006 3:24 pm

Hello,

1. Where would you put a C-terminal HIS tag sequence in a cDNA of interest? (i.e. how close to your stop codon is most favorable).

2. HIS can be transcribed using CAC or CAT. Is it critical to use one versus the other when our aim is to express and purify proteins?

3. If there are two HIS tags, one at the N-terminal and one at the C-terminal, would that create a problem (any problems you might think of)?

Thank you.

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LilKim
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Post by LilKim » Tue Mar 28, 2006 4:26 pm

I've never inserted his tags.... But off the top of my head,

1. i would start off by putting the tag immediately 5' of the stop codon (any further 5' prime could theoretically interfere with translated protein structure.)... or insert it immediately 3' to ATG.

(however, it is possible that the 5' or 3' end amino acids would be tucked and sequestered away from the antibodies in solution ...so if you know anything about the 3D structure use that as an aid ... and put the tag in a more "open" area of the molecule. And, if you don't know much about the structure ... look at the amino acid sequence and you would want to stay away from the "end" that seems more hydrophobic.)

2. Hmm.. i would think that CAC Vs. CAT wouldn't matter?? But i'm don't know if scientist use one over the other conventionally.

3. Why would you want to insert two tags? You should pick one (N or C) ... if you put one on both ends you're going to need twice as many antibodies to pull it out of solution. (cuz you'll have twice as many Antibodies binding to each molecule) Furthermore, the more "stuff" you add to a protien... the less likely your protein will still function normally. (you'd be introducing a source of error.... However! if you want to KILL the function of a protein add TONS of HIS tags)

again.. i've never done HIS tagging... so i may be completely wrong...

good luck!

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canalon
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Post by canalon » Tue Mar 28, 2006 11:05 pm

1- usually beginning or end of the protein. An idea of structure helps, otherwise you do as you want, or as your commercial His-tagging kit goes...

2- I do not think it would really matter, but if you want to be really carefull look for known codon bisases for his in your organism.

3- @Lilkim: His-tag binds to Zn resin, no Ab needed, so that is not a problem.
Adding more tags can create problems for correct folding, so usually it is safer to use only one tag for later use, even if you will be cleaving it after purification.
Patrick

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