Question

His-tagged recombinant antibodies expressed in E. coli

Hello fellow netters,

I have a question for people in field of recombinant antibodies. For some reason, we are unable to get active c-terminal 6x His-tag, when recombinant antibody is expressed in E.coli, confirmed either by ELISA or WB. Protein is expressed in correct length, confirmed by MALDI-TOF. My feeling tells me , this might be due to the c-terminal sequence (Ala-Ala-Ala-Leu-Glu), preceding His-tag, introduced by cloning of an antibody.
Nevertheless there are other publications on net, without problems with active His-tag, directly in frame with c-terminus of antibody. Might be the result of common process, not to publish, what is not working.
If anybody experienced similar issues, please share them with me.

M.
May 30, 2012

Topics

0 / 0  ·  7 Answers  ·  202 Views

All Answers (7)

  • Tomáš Hluska · Palacký University of Olomouc
    Zdravím :)

    I don't think the problem is due to few introduced amino acids. I've heard about problems with detecting His-tag with commercial antibodies, so that could be one reason. The other could be that the His-tag is not accessible for antibody. I guess you have purified your protein on Ni-NTA, right?
    May 30, 2012
  • Matej Zábrady · Masaryk University
    Ahoj,

    inaccessibility is the main reason, therefore even IMAC purification is not working for our molecules. Seem to be a rule of thumb, whether it is accessible or not, given the fact, some papers reported purification/detection of His-tagged scFv. My question was more of an curiosity, if other labs experience similar issue.
    May 30, 2012
  • Claudio Santoro · Università degli Studi del Piemonte Orientale "Amedeo Avogadro"
    are your recombinant antibodies scFvs? try to have them as periplasmic proteins. we usually use sv5 tag but when we had to use his tag (pET -based vectors) we did not face real problems
    May 30, 2012
  • Rebecca Thompson · Pharmasan Labs
    I would agree that accessibility is an issue. For Western blots we always use a NuPAGE Bis Tris gel.
    May 30, 2012
  • Priyabrata Mohanty · Indian Institute of Advanced Research
    Tomáš Hluska is right. Inaccessible is the main reason. I am myself working on transmembrane protein and I find it difficult to find literature of western from lysate until protein is purified. I have faced similar problems too but upon standardisation and using good vector, my problem is solved to some bit.
    May 30, 2012
  • Matej Zábrady · Masaryk University
    @Claudio

    Yes, they are expressed as periplasmic protein (pET22). Changing the His-tag to myc-tag resolved the detection issue, but it is more inconvenient to handle purification without His-tag, you know what I mean. What is the format of your scFv regarding chain order and flanking restriction sites? Do you fuse His-tag directly to c-terminal of an antibody ?
    May 31, 2012
  • Ancy Thomas · Anna University, Chennai
    hi
    There is a possibility that the His tag is hidden in the folds of your protein. U can treat the protein with urea or ant other mild detergents which denature the antibody and expose the hidden grooves and thus the his tag too.... All the best.
    Jun 7, 2012

Question Followers (7)