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xiangao金虫 (正式写手)
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融合基因
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我现在想构建一个融合蛋白基因并且得到表达，刚接触以前没做过，请高手指点一下，需要什么注意事项？ 听说两个融合基因之间有个linker,是这样吗？直接overlap，去掉前一个基因的终止密码子，和后基因的起始密码子，可以吗？ 还有什么注意的啊？ [ Last edited by xiangao on 2009-9-29 at 20:52 ] |
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4楼2009-09-30 08:19:30
mascotte
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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 ] |
3楼2009-09-30 07:29:32
xiangao
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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! |
5楼2009-09-30 10:22:04
mascotte
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6楼2009-09-30 22:40:49
