| | Stoichiometric protein complex formation and over-expression using the prokaryotic native operon structureEdited by Gianni Cesareni Received 20 July 2009; received in revised form 22 December 2009; accepted 22 December 2009. published online 18 January 2010. Abstract In prokaryotes, operon encoded proteins often form protein–protein complexes. Here, we show that the native structure of operons can be used to efficiently overexpress protein complexes. This study focuses on operons from mycobacteria and the use of Mycobacterium smegmatis as an expression host. We demonstrate robust and correct stoichiometric expression of dimers to higher oligomers. The expression efficacy was found to be largely independent of the intergenic distances. The strategy was successfully extended to express mycobacterial protein complexes in Escherichia coli, showing that the operon structure of gram-positive bacteria is also functional in gram-negative bacteria. The presented strategy could become a general tool for the expression of large quantities of pure prokaryotic protein complexes for biochemical and structural studies. 1. Introduction  The structural and functional characterization of multi-component protein complexes is a major area of study in the field of systems biology. In the past decade of the post genomic era, several protein interaction networks of diverse organisms have been reported [1]. These studies have revealed that proteins often do not function in isolation within cells, but form functional complexes by associating to binding partners. Although it has been known for a long time that functionally related genes are organized in operons in prokaryotes and that they are transcribed in a coordinated manner [2], systematic computational studies for prediction of operons have only been carried out recently [3]. To study protein complexes at the structural or biochemical level, generally milligrams of purified complex are required. Recombinant technology has been routinely used to produce single proteins using heterologous expression systems. Although several co-expression strategies are available for both eukaryotic and prokaryotic hosts, it often remains challenging to obtain large amounts of native protein complexes with defined stoichiometric functional compositions [4]. Here we take advantage of the fact that functionally related genes in prokaryotes are organized in operons and that they are co-transcriptionally and co-translationally regulated [2], [5], [6]. Currently about 1110 bacterial genomes have been sequenced [7]. Systematic computational tools for the annotation of genes are being developed and several studies are available for predicting prokaryotic operons [3], [5], [8]. Studies aimed at predicting Mycobacterium tuberculosis (M. tuberculosis) operons have recently suggested that there are more than 1000 operons consisting of two or more open reading frames (ORFs) [3], [9], [10]. These data also indicate that more than half of the 4000 ORFs of M. tuberculosis are organized in operons [3], [11] (Supplementary Table 1). In this study we have investigated the possibility of establishing a generic, effective and reliable expression and purification method for protein complexes by exploiting the native operon structures in M. tuberculosis. Initially we explored whether native operons are functional in a homologous expression host of the same taxon, in this case M. smegmatis. As proof of principle, we explored three paralogous and two orthologous operons of the WXG-100 protein family. To further demonstrate the applicability of our method, we extended the study to the operon encoding the proteasome, a large protein oligomer. Furthermore, we show that the operon structures of a gram-positive bacterium (M. tuberculosis) are also recognized heterologously by a gram-negative bacterium (Escherichia coli). This study represents a substantial advance in that (i) it reduces the design of multi-gene expression constructs to a single step and (ii) it permits the expression of milligrams of protein complex in a native and functional state using inducible promoters. 2. Materials and methods 2.1. Construction, expression and purification of recombinant protein complexes For the construction of the expression vectors, the studied operons were amplified from genomic DNA of M. tuberculosis (strain H37Rv), M. leprae and M. smegmatis (strain mc2 155) using PCR. The employed expression vectors were pMyNT, a pSD26 derivative, for over-expression in M. smegmatis [12] and pETM-Z (a kind gift from Günter Stier) or pETM-11 [13] for over-expression in E. coli. The primers were designed such that the PCR products could be ligated into the expression vectors, which were linearized using the restriction enzymes, NcoI, BamHI and HindIII, respectively. Primers are listed in Supplementary Table 2. All methods relating to protein expression in M. smegmatis were carried out following the reported method [12]. In brief, the M. smegmatis mc2 155 competent cells were transformed with the positive recombinant plasmids using electroporation and plated out on 7H10 agar plates supplemented with 50 μg/mL hygromycin. Single colonies were inoculated to express the protein complex of interest. The culture medium consisted of Middlebrook 7H9 medium supplemented with 0.2% (v/v) glycerol, 0.2 % (w/v) glucose, 0.05% (v/v) tween 80 and 50μg/mL hygromycin. The culturing condition was 37°C with a 200rpm shaking speed. The protein expression was started at 2.0 OD600nm by adding acetamide to a final concentration of 35mM. Expression in E. coli was carried out by following the reported method [32] using BL21(DE3) Codon Plus RIL strain (Stratagene). In both expression systems the cells were incubated over night after induction of protein expression. The purification of the expressed protein complexes was standardized. The crude extracts were obtained by centrifugation (30.000 rcf) after sonication of the cells in the extraction buffer, 50mM Tris–HCl, pH 8.0, 300mM NaCl and 20mM imidazol. The protein complexes were further purified in three steps. First, His-affinity chromatography was performed using 50mM Tris–HCl, pH 8.0, 300mM NaCl, 500mM imidazol as elution buffer. Second, the His-tag was removed by TEV protease treatment in extraction buffer supplemented with 10mM β-mercapto-ethanol, and as the polishing step size-exclusion chromatography was applied using the buffer, 50mM Tris–HCl, pH 8.0, 300mM NaCl. 2.2. Mass spectrometry analysis The purified complexes were identified by mass spectroscopic analysis. The samples were prepared according to the manufacturer’s instruction. In brief, the excised bands from the SDS and native gels were destained, dehydrated, vacuum dried and incubated overnight with methylated porcine trypsin (trypsin gold, Promega). Peptides, as well as full-length complex, were analyzed with MALDI-TOF using a Voyager-DE-STR mass spectrometer. 2.3. Sample preparation for electron microscopy (EM) The specimens for electron microscopy were prepared according to published methods [14]. In brief, about 1–2μL proteasome solution at a concentration of 0.5μM were applied to a carbon coated, glow discharged 600-mesh EM grid. The specimens were negatively contrasted by overlaying with 2% uranyl acetate. The electron micrographs were recorded on a Philips CM12 microscope in low dose mode. 3. Results 3.1. Native operon structures are recognized by bacteria within the same taxon Comparative genomic analyses of M. tuberculosis and the avirulent vaccine strain BCG have revealed 16 Regions of Difference, designated RD1-RD16, respectively [11], [15], [16]. Among them, RD1 is the only region which is present in all virulent strains and absent in all avirulent ones [17], [18]. The potential virulence factors CFP-10 and ESAT-6 have been studied intensively due to their property to generate strong T cell immune responses [19]. They are encoded in an operon that is an integral part of RD1 [20]. CFP-10 and ESAT-6 (Rv3874, Rv3875) form a 1:1 heterodimeric complex [21], [22]. The two proteins are part of a large protein family, which is generally present among mycobacteria irrespective of their virulence [23]. The genome of M. tuberculosis contains 11 CFP-10/ESAT-6-like pairs, five pairs of which are situated within their own gene clusters encoding a type-VII secretion system as in RD1. The NMR structure of the CFP-10/ESAT-6 complex was determined from proteins that were separately expressed in E. coli and co-refolded, post purification, to produce a heterodimeric complex [21]. Here, we have cloned the entire coding region of the CFP-10/ESAT-6 operon, including their intergenic base pairs into the expression vector pMyNT (Fig. 1A), to be used in the homologous expression host M. smegmatis. The CFP-10/ESAT-6 complex could be expressed and purified in an equimolar ratio of the two protein components (Fig. 2A, lanes 1 and 4). The yield of purified protein complex was about 8mg per litre of cell culture. We further tested the method for other CFP-10/ESAT-6-like coding operons, all of which possess different intergenic distances ranging from 10 to 32 base pairs (Fig. 1B). The sequence identity between the different paralogs and orthologs is in the range of 15–65%. We were able to successfully express three CFP-10/ESAT-6 paralogous complexes from M. tuberculosis and the orthologous complexes from M. leprae and M. smegmatis (Fig. 4). All pairs were expressed in equimolar ratios and therefore migrated as single bands with native PAGE (polyacrylamide gel electrophoresis) (Fig. 4B). This confirms that the native operon structures can be used for homologous recombinant expression of targets consisting of two ORFs, irrespective of the different intergenic distances and base pairs. To generalize and to demonstrate the advantages of our method in the context of potentially challenging large protein assemblies, we selected a high molecular weight test system from M. tuberculosis, the proteasome complex, for expression. The proteasome is encoded by an operon containing the ORFs Rv2109c (α-subunit) and Rv2110c (β-subunit), forming an α7β7β7α7 complex with a molecular weight of 730kDa [24]. With our protocol, this proteasome complex could be generated in 10mg quantities from 1L of culture (Fig. 4, lane 7). 3.2. Biophysical and structural biological characterization of the protein complexes All protein complexes, expressed and purified using their native operon content, were analyzed using circular dichroism spectroscopy, static light scattering and mass spectrometry. All proteins of CFP-10/ESAT-6-like complexes show a CD spectrum with high α-helix content (Supplementary Fig. S2) and form a 1:1 stoichiometric complex (Fig. 2, Fig. 4). The analysis using mass spectrometry revealed that all proteins are full-length proteins. The peptide mass fingerprinting analysis using MALDI-TOF spectrometry showed that the proteins co-migrate as one band on a native-PAGE and the band contains the CFP-10/ESAT-6 complex (Fig. 2B, Supplementary Fig. S1). All other protein complexes also co-migrate as one band on a native-PAGE and form 1:1 stoichiometric complexes. To further assess whether the proteins form complexes with functional conformations we analyzed the selected complexes using X-ray crystallography and electron microscopy. We were able to crystallize the CFP-10/ESAT-6 complex of the RD-1 region and determine its structure (Fig. 3A). It forms a tight, four-helical bundle resembling the published NMR structure [25]. The purified proteasome complex was characterized using electron microscopy. It forms a α7β7β7α7 complex with a barrel-like structure (Fig. 3B). 3.3. Native operon structure of M. tuberculosis, a gram-positive bacteria, is recognized by the gram-negative bacteria, E. coli After demonstrating that the native operon structure of mycobacterial genomes can be transferred to a homologous recombinant expression system, we wanted to determine if the method could be generally applied to all prokaryotes, by transferring the operons into the heterologous E. coli expression system, the most used expression system. To establish proof of principle we chose the operon encoding the CFP-10/ESAT-6 protein complex of M. tuberculosis [23], which we studied extensively in this report, for expression in E. coli. The entire coding region of the CFP-10/ESAT-6 operon was cloned into the pETM-Z expression vector. Effectively we replaced the native promoter with the inducible T7 promoter, which controls the transcription of the cloned operon (Fig. 1A, middle and bottom). The expressed complex consists of double-tagged CFP-10 and untagged ESAT-6. The tag consists of an N-terminal His6-tag and a solubilisation Z-tag, a domain of protein A originating from Staphylococcus aureus (Fig. 2A, lane 3). Interestingly, both proteins encoded in the M. tuberculosis operon are expressed in E. coli, demonstrating that the operon structure of a gram-positive bacterium (of the phylum Actinobacteria) is recognized by a gram-negative bacterium (E. coli). However, gel electrophoresis revealed that CFP-10, the upstream gene of the operon in our construct, was expressed at levels more than 10-fold higher than ESAT-6 (Fig. 2A, lanes 3 and 6). Recloning the operon into pETM-11, resulting in the removal of the Z-tag, yielded a similar skewed expression pattern (Fig. 2A, lanes 2 and 5), showing that the non-equimolar expression levels are independent of the tag (Fig. 2A, lanes 2, 3, 5 and 6). 4. Discussion In summary, we present a generic strategy for the expression of prokaryotic protein complexes encoded by genes organized in a single operon structure. We have shown that by maintaining the native operon structure in an expression system, it is possible to produce large amounts of protein complexes assembled in correct stoichiometry. One of the aims of this work was to produce protein complexes from M. tuberculosis for structural studies. It has been reported that 30–50% of all M. tuberculosis targets expressed in E. coli result in the formation of inclusion bodies [26], either because of a non-compatible expression host or because the expressed single protein lacks its essential protein complex partner(s). To date, close to 500 structures from about 200 M. tuberculosis ORFs have been deposited in the Protein Data Bank [27], but only four structures of protein–protein complexes are available: the proteasome, the PE25/PPE41 complex, the toxin–antitoxin complex VapBC-5 and the CFP-10/ESAT-6 complex [24], [25], [28], [29]. These four complexes were produced using three different methods: by co-expression in E. coli using a bi-cistronic expression vector with two separate promoters [24], by the use of a single T7 promoter with an additional synthetic ribosomal binding site between the ORFs [28], [29] or by the generation and purification of individual proteins and subsequent co-refolding [25]. Although the first two complexes could be co-expressed, the employed methods are cumbersome, since the ORFs were dissected and handled individually. The method presented in this paper offers substantial advantages over the methods currently being employed; e.g., the cloning effort is reduced to a single step with no need to manipulate multiple genes. Due to the maintenance of the functional genetic context, complex formation will occur co-translationally, and biologically active protein complexes are formed. To demonstrate further the advantages of this method we chose the M. tuberculosis proteasome complex for expression, because (i) the recombinant production of the proteasome complex from M. tuberculosis has been well documented in the literature [30], and (ii) it is a 28mer complex with a molecular weight of 730kDa. We were able to complete all the steps from cloning to the analysis of the proteasome complex using electron microscopy within nine days. These results demonstrate that this method is well suited for delivering large amounts of highly-pure protein complexes that are required for X-ray crystallography and biochemical studies. Many prokaryotic genomes have been sequenced and tools are currently being developed to predict operons and protein–protein interactions [3]. The methodology presented here opens a new avenue to explore prokaryotic protein complexes and experimentally validate bioinformatics-based predictions of complex formation. We believe this new method will become one of the key tools to exploit the rich and diverse genetic information available at the molecular level, and will enable the study of a broad range of complex systems, such as enzymatic pathways, secretion machineries and transcription complexes. This approach is not limited to complexes encoded by a single operon, but could be extended to a multi-operonic expression construct. It could be used to pursue the experimental study of a bacterium in a system biology context at the molecular level. The ready availability of pure protein complexes in milligram quantities has important implications for fundamental research, as well as for drug target validation and for drug discovery targeting human pathogens, such as M. tuberculosis and M. leprae. Acknowledgements We thank Frederice Gries for technical assistance and Gunter Stier for the gift of pETM-Z. We would like to express our appreciation to the Kaufmann lab (Max Planck Institute for Infectious Biology, Berlin, Germany) for providing the initial pSD26 vector. We would also like to thank the Mandelkow Laboratory (Max Planck Unit for Structural Molecular Biology, Hamburg, Germany) for electron microscope access and Gerard Drewes (Cellzome, Heidelberg) and Matthias Ehebauer for critical reading of the manuscript. This work was supported by the EC Grant ScrIn-Silico (LSHP-CT-012127) to M.W., by the BMBF Grant “X-MTB” (0312992A) to M.W., and by a grant within the BMBF programme Pathogenomik Plus (PTJ-BIO 0313801L) to M.W. Appendix A. Supplementary data Table S1. List of complete ORFs of M. tuberculosis. All intergenic distances and predicted possible operons are listed. Adapted from Roback, P. et al. Nucleic Acids Res 35, 5085-95 (2007) (Roback P, Beard J, Baumann D, Gille C, Henry K, Krohn S, Wiste H, Voskuil MI, Rainville C & Rutherford R (2007) A predicted operon map for Mycobacterium tuberculosis. Nucleic Acids Res 35, 5085-5095.). References [1]. [1]Ceol A, Chatr Aryamontri A, Licata L, Peluso D, Briganti L, Perfetto L, et al. MINT, the molecular interaction database. Nucleic Acids Res. 2010;38:D532–D539.
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European Molecular Biology Laboratory (EMBL), Hamburg Outstation, c/o DESY, Notkestrasse 85, 22607 Hamburg, Germany  Corresponding author. Fax: +49 4089902149.
PII: S0014-5793(10)00042-6 doi:10.1016/j.febslet.2009.12.057 © 2010 Federation of European Biochemical Societies | |
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