Protein Expression and PuriWcation 53 (2007) 404–410 www.elsevier.com/locate/yprep 1046-5928/$ - see front matter  2007 Elsevier Inc. All rights reserved. doi:10.1016/j.pep.2007.01.006 A novel peptide tag for detection and puriWcation of recombinant expressed proteins William T. Jones a,¤ , Dawn Harvey a , Christopher Kirk a , Jasna Rakonjac b , Xiaolin Sun a , Nicky Frearson a , Taha Al Samarrai a a The Horticulture and Food Research Institute of New Zealand Ltd, Private Bag 11 030, Palmerston North, New Zealand b Institute of Molecular Biosciences, Massey University, Private Bag 11 222, Palmerston North, New Zealand Received 14 December 2006, and in revised form 10 January 2007 Available online 19 January 2007 Abstract Peptide tags have proven useful for the detection and puriWcation of recombinant proteins. However cross reactions of antibodies raised to the tag are frequently observed due to the presence of host proteins containing all or parts of the tag. In this report we have iden- tiWed a unique viral peptide sequence, R-tag, that by blast searches is absent from the commonly expression hosts Arabidopsis thaliana, Escherichia coli, Pichia pastoris and mouse myeloma cell NSO. We have prepared monoclonal antibodies to this peptide and conWrmed the absence of this peptide sequence from the above genomes by Western blotting. We have also modiWed protein expression vectors to incorporate this sequence as a fusion tag in expressed proteins and shown its use to successfully purify recombinant proteins by immuno- aYnity procedures.  2007 Elsevier Inc. All rights reserved. Keywords: Peptide tag; R-tag; Expression vectors; ImmunoaYnity puriWcation; Monoclonal antibody; Single chain antibody fragment; RGL-2; Expression hosts Expression of recombinant proteins in a variety of expression hosts has become an important approach to characterize gene products for post-genomic research. Alternative systems, such as bacteria, yeast, virus, plant and mammalian cells, for expression of proteins are nec- essary to obtain soluble, correctly folded and appropri- ately translational processed proteins similar to their “native” condition. Such recombinant proteins are required for production of antibody reagents, for func- tional assays, to characterize their native structure and for the development of aYnity reagents to identify inter- acting proteins. Generally these recombinant proteins require puriWca- tion from host cell proteins. To aid in the puriWcation steps, several diVerent tags have been developed and described [1]. These tags have been placed at either the N- or C-terminus of the recombinant proteins and have been used to enhance the solubility of the protein i.e. maltose binding protein [2] and glutathione-S-transferase [3] or for puri Wcation and detection i.e. small peptide tags such as polyhistidine [4], FLAG [5] and the sequence TKDPSRVG to which polyol-responsive MAbs have been prepared that allow antigens to be eluted from the aYnity matrix under mild non-denaturing conditions [6]. Problems frequently arise due to the short peptide tag occurring wholly or in part in proteins present in the expression host organism and resulting in co-puri Wcation or detection of cross-reacting proteins. In this report we present results which use a novel pep- tide, R-tag, that is, by Western blotting, undetected in the commonly used protein expression hosts Escherichia coli [7], Arabidopsis thaliana [8], Pichia pastoris [9] or mouse myeloma cell line NSO [10] using high aYnity monoclo- nal antibodies reactive to R-tag. We also report the puri Wcation, using immunoaYnity chromatography, of * Corresponding author. Fax: +64 6 3517031. E-mail address: wjones@hortresearch.co.nz (W.T. Jones).