insert small peptide - (Oct/17/2005 )

Dear all,
I got a problem here. I have my targeted gene already inserted into a vector. but now I want an extra histag at the end of my protein. how can I insert such a small frgment about 15bp into my vector at the N-terminal of my gene.

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Not to burst your bubble, but this maybe a problem. If you have a retsriction site directly upstream of the start codon, theoretically you could synthesize the HA tag with oligos, adding the restiction sites and the 5' and 3' ends, and clone it up stream of the open reading frame. You need to verify that the fusion protein will be in frame. By inserting an extra base or two you maybe able to "correct" the reading frame and have it expressed properly. In this case you may have to add another amino acid such as alanine just to have it expressed properly. Good luck!

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hi
i would do a PCR, with a prier allowing the epitope tagging, and the other primer would end at a restriction site in the protein. Then digestion of both and ligation with a bigger fragment.
Easier is to reclone the ORF in a tagging vector (such as pFHM, or similar).
fred

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I can not change the vector because the vector I am using contain a MBP tag. drive me mad about these things. after i go through all around, now the MBPtagged protein yield is quite low and I need to cleave off the MBP and get rid of the protease. so after all these procedure I guess I will lost all proteins. so now I want to change the strategy, add an extra His tag, so I can purify it easily with high yield. now seems quite hard.

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Kind of hard to figure out without all the details, but sounds like you have to reclone this and start over.

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if i understand, youn have (RS is for restriction site)
promoter RS1 / MBP / RS2 / ORF / RS3 ?

So hat about doing a pcr with 5' oligo having RS1 / HIS / RS what you want / ORF...
and the 3' oligo : RS3 / ORF ?

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now I have my Hind III-ORF-TEv cleavage site--Bam Hi-MBP-NdeI-promoter
and I want to change to 6xHis-MBP-TEV cleavage site-MY ORS-Promoter
is it clear? by the way, the longest primer could be how long?
Generally 6xHis, is it 5xHis will work too?

Thank you guys so much.

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TEv cleavage site--Bam Hi-MBP-NdeI-promoter
MBP-TEV cleavage site-MY ORS-Promoter

i don't get that change clear...

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so basically the change is except add an extra 6xHIS, i change the position of the MBP tag and my protein. so my protein is nearest to the promoter, so if my protein is not translated completed, the MBP tag is not going be translated. so i will not get truncated protein with tag. all protein with the tag will be full length of my protein. is that clear?

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TEv cleavage site--Bam Hi-MBP-NdeI-promoter
MBP-TEV cleavage site-MY ORS-Promoter

i've understood by this that you want to change the direction of the orf?... or what is ORS?...

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