205 Nikolaos E. Labrou (ed.), Protein Downstream Processing: Design, Development and Application of High and Low-Resolution Methods, Methods in Molecular Biology, vol. 1129, DOI 10.1007/978-1-62703-977-2_18, © Springer Science+Business Media, LLC 2014 Chapter 18 An Orthogonal Fusion Tag for Efficient Protein Purification Johan Nilvebrant, Mikael Åstrand, and Sophia Hober Abstract Protein fusion tags are important tools in research when robust methods for protein purification and detection are required. In this chapter we present an efficient method for stringent protein purification. A small domain, denoted ABDz1, with affinity for both human serum albumin and Protein A has been developed. The purification tag is based on an albumin-binding domain from Streptococcal Protein G that was engineered to bind Protein A. The ABDz1-tag can be fused to any protein of choice and the purifica- tion can be performed using standard laboratory equipment. In this chapter a method for purification of ABDz1-tagged proteins using two successive affinity purification steps is described. Key words Orthogonal affinity purification, ABDz1, Protein G, Human serum albumin, Protein A, Albumin binding, Fusion tag 1 Introduction Technologies to enable recombinant protein production have greatly evolved during the last few decades. Those advances have led to improved yields and throughput in the production of a large range or proteins with vastly different origins and characteristics. A number of different methods for purification of recombinantly produced proteins have also been developed to meet the demands set by researchers and industry. To achieve high purity with few unit operations, affinity chromatography using one of many avail- able fusion tags has become a common method of choice. One example of a frequently used affinity tag is the His 6 -tag [1] used in immobilized metal ion affinity chromatography (IMAC), which remains the standard purification procedure for many researchers [2]. However, more specific and high affinity chromatographic methods may be required to obtain higher purity [3]. To meet the requirements put on protein purification pro- cesses, we have developed a small, 46 amino acid protein domain with affinity to both human serum albumin (HSA) and Protein A [4]. The developed protein, denoted ABDz1, is based on one of