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xiangao

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[ Last edited by xiangao on 2009-9-29 at 20:52 ]
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mascotte

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amisking(½ð±Ò+3,VIP+0):ÎÒ¾õµÃÖÐÎıȽÏÈÝÒ×ÈÃÈË¿´¶®£¡ 9-30 08:27
xiangao(½ð±Ò+5,VIP+0):it's very useful,thank you 9-30 09:36
In your design, have you thought about including a protease site to remove the tag if necessary? By doing this, you also add a little spacer between your protein.

If possible, I was taught to remove the Met for N-terminal fusions, as internal Met are usually rare. I usually just remove the Met and start the coding sequence of protein with the second amino acid, I do not replace the Met with another amino acid.  I don't think the addition of one amino acid is going to change things. I mean, is there any real difference between the last amino acid of your tag and a residue added to replace the Met?

If you are set on adding an amino acid, I would choose something neutral, like alanine or possibly valine (glycine or leucine could work as well). It should be noted, in bacteria, Val (GTG) can act as a start codon. I'm not sure proline would be good, since it is not very flexible and can cause kinks in the protein structure.

For larger fusions, for example GFP, I have seen people make flexible spacers consisting of Ser/Thr-Gly repeats. I believe it looks something like S-G-G-G-G-S. You can add these units in tandem for larger spacers if needed.

Finally, you can always look at commercial expression vectors to see the kinds of amino acids they use as spacers between different elements.

[ Last edited by mascotte on 2009-9-30 at 08:04 ]
3Â¥2009-09-30 07:29:32
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bioxixi

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xiangao(½ð±Ò+1,VIP+0):¸Õ¿ªÊ¼×ö£¬ÄãдµÄ¿Ï¶¨¶ÔÎÒ¶¼ÓаïÖú 9-30 11:19
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4Â¥2009-09-30 08:19:30
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xiangao

½ð³æ (ÕýʽдÊÖ)

ÒýÓûØÌû:
Originally posted by mascotte at 2009-9-30 07:29:
In your design, have you thought about including a protease site to remove the tag if necessary? By doing this, you also add a little spacer between your protein.

If possible, I was taught to re ...

thank you for your reply.I wonder if a cut is not necessary between the tag and target protein, can I get an active protein? It is said most of the fusion protein is deactive,maybe because the former protein repress or hinder the active site where the substrate and enzyme react
can you recommend some related papers or literature about this work to me?
wish your guide!
5Â¥2009-09-30 10:22:04
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mascotte

½ð³æ (ÕýʽдÊÖ)

some fusion protein may lost part of their function but not all. as an example, the combination may affect the ability of entering nucleus. But you can still use GFP fusion protein to detect their location, use GST fusion protein to pull down.
6Â¥2009-09-30 22:40:49
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