Functional genomics in Trypanosoma brucei: A collection of vectors for the expression of tagged proteins from endogenous and ectopic gene loci

Abstract

The expression of transgenes encoding proteins modified to contain residues that impart a particular property, ‘tagged proteins’, is central to the post-genomic analysis of any organism. Trypanosoma brucei is a model kinetoplastid protozoan pathogen and has the most advanced repertoire of tools for reverse genetic analysis available for any protozoan. The vast majority of these tools take advantage of the predominance of homologous over non-homologous recombination to target constructs to specific genomic loci. Initially the targeting was used to direct unregulated transgenes to transcribed regions of the genome [1] and to perform gene deletions [2,3]. A leap forward in the sophistication of reverse genetic experiments occurred with the development of trypanosome cell lines expressing the tetracycline repressor (TetR) protein which facilitated tetracycline-regulated expression of transgenes [4,5]. Further development of cell lines expressing both TetR and bacteriophage T7 RNA polymerase (T7RNAP) allowed transgenes to be transcribed and expressed at very high levels [6]. The TetR- and T7RNAP-expressing cell lines are also central to most RNA interference-based analyses of gene function currently performed in trypanosomes [7–10]. In nearly all cases, an antibiotic resistance gene is used as the selectable marker. The DNA used for targetted integration is usually a linearised plasmid; targetting using PCR products directly is possible [11–13] but integration is less efficient and does not usually offer inducible expression. The expression of tagged proteins has become central to the technologies that have developed to analyse the function of individual genes. The tag can have a range of functions falling into two main categories: the first is to provide evidence for the sub-cellular localisation of a protein in living cells using a fluorescent protein tag (see, for example [14]) or in fixed cells using a fluorescent protein or epitope tag (see, for example [15]). The second is to facilitate the purification of complexes which, when allied with mass spectrometric analysis and knowledge of the genome sequence, allows the identification of components of multimeric proteins. Two types of tag have been developed successfully in yeast: (i) the tandem affinity tag where two successive rounds of purification are used [16], and (ii) a tandem epitope tag which is used in a single step purification [17]. To date, the former has been exploited more in trypanosomes [18,19]. In the future, the investigation of transient protein-protein interactions in vivo could be analysed by techniques such as fluorescence resonant energy transfer (FRET) which is dependent on tagging the two target proteins with different fluorescent proteins [20]. Here, we describe five sets of plasmids that represent a substantial collection of publicly available vectors for adding tags to the N- and C-termini of proteins in T. brucei.