A double epitope tag for quantification of recombinant protein using fluorescence resonance energy transfer.
The expression of recombinant proteins is a well-accepted technology, but their detection and purification often require time-consuming and complicated processes. This paper describes the development of a novel double epitope tag (GEPGDDGPSGAEGPPGPQG) for rapid and accurate quantification of recombinant protein by a homogeneous immunoassay based on fluorescence resonance energy transfer. In our double epitope tagging system, recombinant proteins can be simply measured on a microtiter plate by addition of a pair of fluorophore-labeled monoclonal antibodies (their epitopes; GEPGDDGPS and GPPGPQG). The sensitivity of the immunoassay with an incubation time of only 5 min is almost equal to that of labor-intensive Western blotting. In addition, culture media and extracts of host cells generally used for protein expression have little effect on this immunoassay. To investigate the utility of our proposed tag for protein production, several different proteins containing this tag were practically expressed and purified. The data presented demonstrate that the double epitope tag is a reliable tool that can alleviate the laborious and troublesome processes of protein production.
Enomoto
Koji
K
Discovery Research Laboratories, Shionogi & Co., Ltd., 5-12-4, Sagisu, Fukushima-ku, Osaka 553-0002, Japan.
Uwabe
Ken-Ichiro
K
Naito
Shoichi
S
Onoda
Jyunji
J
Yamauchi
Akira
A
Numata
Yoshito
Y
Takemoto
Hiroshi
H
D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal / Q000096:biosynthesis / Q000737:chemistry / Q000276:immunology; D015153:Blotting, Western; D024043:Collagen Type II / Q000276:immunology; D000939:Epitopes / Q000032:analysis / Q000737:chemistry*; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D006801:Humans; D007118:Immunoassay / Q000379:methods; D007128:Immunoglobulin Fragments / Q000032:analysis / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D008969:Molecular Sequence Data; D009942:Organometallic Compounds / Q000737:chemistry; D010455:Peptides / Q000737:chemistry* / Q000276:immunology; D011994:Recombinant Proteins / Q000032:analysis* / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D011436:Spermidine Synthase / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics
D000911:Antibodies, Monoclonal; D024043:Collagen Type II; D000939:Epitopes; D007128:Immunoglobulin Fragments; D009942:Organometallic Compounds; D010455:Peptides; D011994:Recombinant Proteins; C070734:europium(III) trisbipyridine cryptate; D011436:Spermidine Synthase