Posted 28 November 2004 - 11:31 PM
I want to link a peptide to the C-term of a protein. Should I mutate the C-term to suit the HindIII N-term of the peptide, or the HindIII (which translates to Lys-Leu ) is not a suitable linker and might interfere with the folding of the original protein?
Is there any basic rule when choosing linkers or is it on a case-by-case basis?
Thank you in advance.
Posted 29 November 2004 - 02:54 AM
Linker not only affect folding of fusion protein but also can react/bind with other proteins. It is became serious problem if the protein that you want to produce is antibody. Be carefull about it. Based on my experience, the use of small linker (for examper His6) didn't affect folding or binding (in vitro).
Posted 29 November 2004 - 09:06 PM
Thanks for the suggestion. The protein is GFP actualy. You are using His you say. Now that I checked, I see that many people use His6 linkers. I wonder why though. Do you know?
Also how do you do the His linker? Is there a commercial way, or I just need to manipulate the coding sequence?
Thanks and all the best,
Posted 30 November 2004 - 07:57 PM
His linkers can be added to the sequence of your interest in a simple way, There are commercial vectors available where the palsmid it self will have the His6 sequences. you can just insert your sequence in such a way that His-tag sequences come either at c-terminus or n-terminus site of your target sequence.
A minimum of 6 Histidine residues are needed for the affinity purification of you His- tagged protein on Ni+ column.
his6-tag vectors i used are pQE 60 and pET 28A+