Affinity fusion strategies for detection, purification, and immobilization of recombinant proteins.

Genetic engineering enables the construction of gene man Genome Project (5), efficient and robust production and purification strategies are necessary (6). For fusions resulting in fusion proteins having the combined properties of the original gene products. Fusion industrial production of recombinant proteins, simple and fast purification methods introduced as an early can be made on either or both sides of the target gene depending on the specific application, but the majority unit operation can improve the overall economy of the process. A powerful purification technique made possiof the described fusion protein systems place the protein of interest at the C-terminal end of a well-characble by the introduction of genetic engineering is to purify the target protein by the use of a genetically fused terized fusion partner. Many different applications for fusion proteins in widespread areas of biotechnology affinity fusion partner. Such fusion proteins can often be purified to near homogeneity from crude biological have been reported (1–3), including a facilitated purification of the target protein, means to decrease protemixtures by a single, and fusion-partner-specific, affinity chromatography step. olysis of the target protein, display of proteins on surfaces of cells and phage, and construction of reporter To date, a large number of different gene fusion systems, involving fusion partners that range in size from molecules for the monitoring of gene expression and protein localization as well as to increase the circulaone amino acid to whole proteins capable of selective interaction with a ligand immobilized onto a chromation half-life of protein therapeutics. However, as pioneered by Uhlen and co-workers in 1983 (4), the most tography matrix, have been described. In such systems, different types of interactions, such as enzyme–subfrequent application of gene fusions has been for the purpose of affinity purification of recombinant proteins. strate, bacterial receptor–serum protein, polyhistidines–metal ion, and antibody–antigen, have been utiThis review focuses on the use of affinity tag fusions for detection, purification, and immobilization of relized (7). The conditions for purification differ from system to system and the environment tolerated by the combinant proteins. A general overview of commonly used affinity tags is followed by some recent examples target protein is an important factor for deciding which affinity fusion partner to choose. In addition, other facof specific applications for affinity tags and a discussion concerning possible future trends. tors, including protein localization, costs for the affinity matrix and buffers, and the possibilities of removing the fusion partner by site-specific cleavage, should also AFFINITY FUSION PARTNERS be considered. Numerous gene fusion systems for afThere is a great interest in developing methods for finity purification have been described in the literature, fast and convenient purification of proteins. For examall with different characteristics (3,7–10). Some of the ple, to facilitate functional and structural studies of most commonly used systems are listed in Table 1, toproteins derived from the rapidly growing number of gether with their respective elution condition(s) and genes coming out of genome programs such as the Huagents suitable for specific detection of expressed fusion proteins. The introduction of a recognition sequence for a 1 Present address: Pharmacia and Upjohn AB, Department of Biochemical agent or a protease between the fusion parttechnology, S-11287 Stockholm, Sweden. ner and the target protein allows for site-specific cleav2 To whom correspondence should be addressed at Department age of the fusion protein to remove the affinity fusion of Biochemistry and Biotechnology, Kungliga Tekniska Hogskolan, Teknikringen 30, S-100 44 Stockholm, Sweden. partner (see below). However, for many applications