Analytical Biochemistry 346 (2005) 327–329 www.elsevier.com/locate/yabio 0003-2697/$ - see front matter  2005 Elsevier Inc. All rights reserved. doi:10.1016/j.ab.2005.06.016 ANALYTICAL BIOCHEMISTRY Notes & Tips ModiWcation of recombinatorial cloning for small aYnity tag fusion protein construct generation Pawel Listwan a,b,c,d,¤ , Nathan Cowieson b , Mareike Kurz b , David A. Hume a,b,c,d , Jennifer L. Martin a,b,c,d , Bostjan Kobe a,b,c,d a School of Molecular and Microbial Sciences, University of Queensland, Brisbane, Queensland 4072, Australia b Institute for Molecular Bioscience, University of Queensland, Brisbane, Queensland 4072, Australia c Cooperative Research Centre for Chronic InXammatory Diseases, University of Queensland, Brisbane, Queensland 4072, Australia d Special Research Centre for Functional and Applied Genomics, University of Queensland, Brisbane, Queensland 4072, Australia Received 11 April 2005 Available online 29 June 2005 Here we present a modiWcation of the recombination reaction (BP reaction) of the Gateway technology cloning procedure (Invitrogen, Carlsbad, CA) that allows expres- sion of fusion proteins containing small aYnity tags such as hexahistidine tags (His-tag), 1 without translation of the nonnative amino acids of the recombination sites. This method is applicable to high-throughput applications, as the PCR products that are generated in this method can be used for the construction of fusion proteins with diVerent N- and C-terminal aYnity tags with the use of a single pair of adapter primers. Structural and functional genomics initiatives have directed great eVort toward improvements in the areas of automation and the design of high-throughput tools for recombinant protein production [1]. One of the biggest bot- tlenecks is the production of soluble proteins, especially where the target proteins are of mammalian origin. The reasons for this include the lack of relevant posttransla- tional modiWcations and folding chaperones in heterolo- gous hosts such as bacteria, frequently resulting in the production of insoluble proteins [2]. One way to increase the success rate of producing soluble proteins in bacteria is the incorporation of protein fusion tags such as glutathi- one-S-transferase or maltose-binding protein, which simul- taneously facilitate protein folding and aYnity puriWcation [3]. Smaller tags, such as the His-tag, oVer the advantage that they usually do not need to be removed from the expressed protein product for functional and structural studies. Many cloning strategies are available for the construc- tion of expression vectors with the desired fusion tag. In conventional cloning, a restriction enzyme map of each cDNA needs to be carefully analyzed so that appropriate sites are chosen that match both the target and the vector DNA. A more recent method, ligation-independent cloning (LIC), does not require the use of restriction enzymes, DNA ligase, or alkaline phosphatase [4]. It is well suited to automation [5,6] but is applicable only to bacteria as a host. Commercially available LIC vectors can incorporate diVer- ent aYnity puriWcation tags and protease (e.g., thrombin, enterokinase, factor Xa) cleavage sites. The system can be modiWed easily to include diVerent protease cleavage sites such as tobacco etch virus (TEV) protease [6]. The bacteriophage lambda-based site-speciWc recombi- natorial cloning represents a new advance in cloning tech- niques [7]. The commercial development of this methodology, the Gateway system (Invitrogen), works via the initial generation of an entry vector construct and sub- sequent transfer into multiple expression (destination) vec- tors, with a choice of bacterial, baculovirus, or mammalian expression hosts [8]. All transfers are achieved without the need for analysis of the restriction sites or the use of restric- tion enzymes [9]. An entry construct can be generated in two ways. The Wrst possibility is the directional topoisomer- ase-mediated cloning (TOPO cloning) [10] that requires the inclusion of four additional nucleotides (CACC) in the gene-speciWc sense primer. The resulting PCR products are cloned directionally by recognizing the GTGG overhang in * Corresponding author. Fax: +61 7 3365 4699. E-mail address: listwan@uq.edu.au (P. Listwan). 1 Abbreviations used: His-tag, hexahistidine tag; LIC, ligation-indepen- dent cloning; TEV, tobacco etch virus; TOPO, topoisomerase.