Abstract
Affinity tag systems are an essential tool in biochemistry, biophysics, and molecular biology. Although several different tag systems have been developed, the epitope tag system, composed of a polypeptide “tag” and an anti-tag antibody, is especially useful for protein purification. However, almost all tag sequences, such as the FLAG tag, are added to the N- or C-termini of target proteins, as tags inserted in loops tend to disrupt the functional structure of multi-pass transmembrane proteins. In this study, we developed a novel “RIEDL tag system,” which is composed of a peptide with only five amino acids (RIEDL) and an anti-RIEDL monoclonal antibody (mAb), LpMab-7. To investigate whether the RIEDL tag system is applicable for protein purification, we conducted the purification of two kinds of RIEDL-tagged proteins using affinity column chromatography: whale podoplanin (wPDPN) with an N-terminal RIEDL tag (RIEDL-wPDPN) and human CD20 with an internal RIEDL tag insertion (CD20-169RIEDL170). Using an LpMab-7-Sepharose column, RIEDL-wPDPN and CD20-169RIEDL170 were efficiently purified in one-step purification procedures, and were strongly detected by LpMab-7 using Western blot and flow cytometry. These results show that the RIEDL tag system can be useful for the detection and one-step purification of membrane proteins when inserted at either the N-terminus or inserted in an internal loop structure of multi-pass transmembrane proteins.
Highlights
Protein purification and detection are essential techniques for pro tein research, including for the determination of structure and elucida tion of function [1]
We first selected a monoclonal antibody (mAb), LpMab-7, the epitope of which was previ ously identified as the pentapeptide Arg79, Ile80, Asp81, Glu82, and Leu83 (RIEDL) [16] from our original mAbs library [17]
We performed the purification of RIEDL-tagged protein using an LpMab-7-Sepharose column to investigate whether the RIEDL tag system can be applied to protein purification
Summary
Protein purification and detection are essential techniques for pro tein research, including for the determination of structure and elucida tion of function [1]. Protein tags, including glutathione S-trans ferase (GST) [5], green fluorescent protein (GFP) [6], and maltose binding protein (MBP) [7], are useful for soluble protein expression; these tags can affect the functions of a target protein, as these tags have high molecular weights Epitope tags, such as FLAG [8], HA [9], Myc [10], and PA [11], are composed of a short polypeptide, which acts as the tag, and are recognized using anti-tag antibodies that exhibit high specificity because of the antigen-antibody interaction. Materials and methods tag, which is recognized by an anti-RIEDL tag mAb (LpMab-7), was subcloned into a pCAG-Neo vector (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan) using the In-Fusion HD Cloning Kit (Takara Bio, Inc., Shiga, Japan)
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