How to design his-tag primer? Do I need to design it at both directions, forward and reverse?
In my case, I have specific primer (F&R) for specific full-length gene. The problem happened when I want to get desired protein expressed from that specific full-length gene in yeast expression system (e.g k. lactis and s. cerevisiae). So far, I can not get any positive signal from enzymatic assay of that expected specific protein. Thus, I choose to design his-tag primer to minimize the error of expressed undesired protein in yeast expression system.
Is it better for me to design his-tag primer rather than just purchasing cloning vector that has already engineered his-tag sequence?
The basic way of HIS-tagging your protein of interest is to include 6 histidine tags either at N terminus or C terminus of protein. If you want to put this tag at N terminal region of protein use this sequence "CATCATCACCACCACCAT " after ATG (i.e. start codon of the gene) in forward primer, if you want to tag at C terminal region of the protein use the antisense strand of above mentioned sequence and add that requence in reverse primer just before the stop codon of the gene. Tagging a protein at N or C terminal region enables us to detect by Western blotting, and for that you dont have to tag at both the sides of the protein, one tag at either side of protein is sufficient.
The basic way of HIS-tagging your protein of interest is to include 6 histidine tags either at N terminus or C terminus of protein. If you want to put this tag at N terminal region of protein use this sequence "CATCATCACCACCACCAT " after ATG (i.e. start codon of the gene) in forward primer, if you want to tag at C terminal region of the protein use the antisense strand of above mentioned sequence and add that requence in reverse primer just before the stop codon of the gene. Tagging a protein at N or C terminal region enables us to detect by Western blotting, and for that you dont have to tag at both the sides of the protein, one tag at either side of protein is sufficient.
thanks for the answer. Can you suggest me the appropriate software to design his-tag primer?
Maybe it can help you,
http://www.genscript.com/his_tagged_protein_detection_purification.html
and to desing a his-tag primer problably it could help you serial cloning, and primer 3
but only you need to add codons of histidine in a side of the sequence.
You can use 5'-CAT CAC CAT CAC CAT CAC-3' as a His tag sequence in any of N terminus or C terminus of gene sequnce. Imidazole competes with the his-tag for binding to the metal-charged resin and thus is used for elution of the protein from an Immobilized metal affinity chromatography (IMAC) IMAC column. And you can purify expressed protein from pichia pastoris using fast protein liquid chromatography (FPLC), is a form of liquid chromatography that is often used to analyze or purify mixtures of proteins.After that you can use Western blotting technique for most purify and specific protein.
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