does 5 his tag matter? - (Dec/16/2008 )

my protein is fusion with his tag , which is from the vector. But after dna sequencing i found only 5 his-tag there, it is supposed to be 6 there. i wonder if it is possible that it is a mistake, but i have 3 same vectors,2 of which have 5 histag and only one has 6 histag. and my protein sequence in the one with 6 histag is wrong, there is a gap in it. So now i wonder if 5 histag is ok for ni-column purification? if it is ok i will just use the one with correct protein sequence and 5 histags. Thanks in advance.

hmm.... this is not my field. However I have noted that there are some systems that do use 5xhis tags. So I would guess that your protein could be purified with a ni-column. Although I would also guess that the binding affinity to the column would be decreased.

Pernesblue is right. You will have slightly weaker binding, so your washing will not be as stringent, so you'll probably elute more contaminants. I'd try eluting with a linear gradient.
The original His tag system used 3-His tags, so your situation is far from hopeless. You're just going to have to do a bit more work.

thank you. But I really wonder why that could happen? There are 6 histags on the pet20+ vector, why it reduces to be 5 histags after I ligate my dna into the vector?

might you have used the XhoI restriction site (which is right next to the his-tag sequence) in the ligation reaction.

If so, the damage to the his-tag would be caused by improper ligation. I have observed that on the rare occasion, the ligation reaction can skip a few bps, ignoring part of the complementary overhang.

If this is not the case, well... it could be mistake in the pET20 plasmid... or some strange mix with a contaminating nuclease in the ligation reaction.