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ETRAP ( e fficient tr apping a nd p urification) of target protein polyclonal antibodies from GST–protein immune sera 1
Author(s) -
Crimmins Dan L.,
Brada Nancy A.,
Lockwood Christina M.,
Griest Terry A.,
Waldemer Rachel J.,
Cervinski Mark A.,
Ohlendorf Matthew F.,
McQuillan Jay J.,
Ladenson Jack H.
Publication year - 2010
Publication title -
biotechnology and applied biochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.468
H-Index - 70
eISSN - 1470-8744
pISSN - 0885-4513
DOI - 10.1042/ba20100279
Subject(s) - immunogen , polyclonal antibodies , affinity chromatography , antibody , recombinant dna , epitope , protein tag , biology , escherichia coli , target protein , protein purification , microbiology and biotechnology , monoclonal antibody , protein g , cloning (programming) , glutathione , myc tag , biochemistry , fusion protein , enzyme , gene , immunology , computer science , programming language
Recombinant GST (glutathione transferase) proteins are widely used as immunogens to generate polyclonal antibodies. Advantages of using GST proteins include: commercially available cloning vectors, vast literature for protein expression in Escherichia coli , the ease of protein purification, immunogen can be used as an ELISA standard and GST can be removed in some systems. However, there are disadvantages: GST oligomerization, inclusion body formation and target protein insolubility after GST removal. Perhaps the most detrimental is the significant generation of anti‐GST antibodies by the host animal. A two‐column procedure using a glutathione‐GST column and a glutathione‐(GST–protein) column can yield affinity‐purified anti‐(GST–protein) polyclonal antibody. Several passes over the first column are often required, though, to completely extract the anti‐GST antibodies from the immune sera. We reasoned that knowledge of the target protein linear epitope(s) would allow construction of a peptide affinity resin for a single‐pass ‘one and done’ purification termed ETRAP ( e fficient tr apping a nd p urification). In the present paper, we describe our efforts and present data on rabbits and sheep immunized with GST proteins having target protein molecular masses of ˜8, 21 and 33 kDa. The titre and purity of the target antibodies using the ETRAP protocol were comparable to the more laborious multi‐column purifications but with a considerable saving in time.