A highly efficient pipeline for protein expression in Leishmania tarentolae using infrared fluorescence protein as marker
© Dortay and Mueller-Roeber; licensee BioMed Central Ltd. 2010
Received: 9 February 2010
Accepted: 10 May 2010
Published: 10 May 2010
Leishmania tarentolae, a unicellular eukaryotic protozoan, has been established as a novel host for recombinant protein production in recent years. Current protocols for protein expression in Leishmania are, however, time consuming and require extensive lab work in order to identify well-expressing cell lines. Here we established an alternative protein expression work-flow that employs recently engineered infrared fluorescence protein (IFP) as a suitable and easy-to-handle reporter protein for recombinant protein expression in Leishmania. As model proteins we tested three proteins from the plant Arabidopsis thaliana, including a NAC and a type-B ARR transcription factor.
IFP and IFP fusion proteins were expressed in Leishmania and rapidly detected in cells by deconvolution microscopy and in culture by infrared imaging of 96-well microtiter plates using small cell culture volumes (2 μL - 100 μL). Motility, shape and growth of Leishmania cells were not impaired by intracellular accumulation of IFP. In-cell detection of IFP and IFP fusion proteins was straightforward already at the beginning of the expression pipeline and thus allowed early pre-selection of well-expressing Leishmania clones. Furthermore, IFP fusion proteins retained infrared fluorescence after electrophoresis in denaturing SDS-polyacrylamide gels, allowing direct in-gel detection without the need to disassemble cast protein gels. Thus, parameters for scaling up protein production and streamlining purification routes can be easily optimized when employing IFP as reporter.
Using IFP as biosensor we devised a protocol for rapid and convenient protein expression in Leishmania tarentolae. Our expression pipeline is superior to previously established methods in that it significantly reduces the hands-on-time and work load required for identifying well-expressing clones, refining protein production parameters and establishing purification protocols. The facile in-cell and in-gel detection tools built on IFP make Leishmania amenable for high-throughput expression of proteins from plant and animal sources.
Leishmania tarentolae is a protozoon of the genus Trypanosoma, and a parasite of the gecko Tarentolae annularis. It has been established as a new eukaryotic expression system for recombinant protein production . An interesting feature of proteins produced in Leishmania is their animal-like N-glycosylation pattern, as demonstrated for erythropoietin . Systems for constitutive and regulated expression of heterologous proteins have been developed [1–3]. Compared to mammalian cell cultures, Leishmania has the advantage of a higher specific growth rate although cultivation in high cell densities (>2 × 108 cells/mL) with a high specific growth rate in serum-free medium was not possible initially. Recently, however, Fritsche et al. developed an alternative growth medium containing hemin, an iron-containing porphyrin essential for growth of Leishmania tarentolae, as the only animal ingredient . Hemin has been shown to stimulate cell proliferation and protein synthesis in L. donovani .
Since its introduction as a new host for protein production which benefited from the development of methods for trypanosomatid cultivation and their genetic manipulation , Leishmania tarentolae has been used for the successful expression of various heterologous proteins such as e.g. proprotein convertase 4 (a member of Ca2+-dependent mammalian subtilases), human laminin-332 and a tissue type plasminogen activator [7–9]. Recently it was also shown that extracts generated from Leishmania cells can be used for protein expression in vitro; in ideal cases up to 300 μg/mL of recombinant protein could be produced within 2 h . However, successful expression of plant proteins in Leishmania cells or in vitro extracts has to our knowledge not been reported so far.
Foldynová-Trantirková et al. have reported a protocol for cost-effective amino-acid-type-selective isotope labeling of proteins expressed in Leishmania tarentolae . The method is based on cultivation of Leishmania cells in a relatively cheap complex medium supplemented with labeled amino acids. The procedure avoids expensive synthetic media.
Although Leishmania has been shown to be a suitable host for foreign protein expression, only a limited number of labs have so far established routine culture and expression pipelines for this organism. A major reason for this may be the lengthy procedure that is normally required to find good expressor clones. The presently suggested protocols for expression of recombinant proteins in Leishmania require a stepwise scale-up of the culture volume before a protein of interest can be detected among several randomly selected clones (for details see manual of the LEXSYcon2 Expression Kit offered by commercial supplier Jena Bioscience; http://www.jenabioscience.com). A typical scheme for setting up protein expression in Leishmania requires seven to eight days.
Recently, an infrared fluorescing protein (IFP) has been engineered as a new reporter protein, derived from a bacterial (Deinococcus radiodurans) phytochrome . IFP covalently binds biliverdin, a natural product of heme catabolism involved in aerobic respiration, and becomes infrared fluorescent with excitation and emission maxima at 684 nm and 708 nm, respectively. Successful expression of IFP has been reported for E. coli, human embryonic kidney cells (HEK293A) and mice. As infrared light relatively well penetrates animal tissue, IFP is suitable for whole-body imaging with negligible background signal, as shown by visualization of liver-expressed IFP in intact mice .
Here we demonstrate that IFP can be employed as a suitable and ease-to-handle reporter protein in Leishmania. We developed a procedure that shortens the currently available protocol for protein expression in Leishmania and significantly reduces overall work load. The newly established work-flow qualifies for multi-parallel protein expression in Leishmania and thus has the potential to be employed in genomics and proteomics research for the functional analysis of proteins.
Biliverdin was purchased from Toronto Research Chemicals (North York, Ontario, Canada), and biliverdin hydrochloride was obtained from Frontier Scientific (Carnforth, Lancashire, UK). Hemin was ordered from Sigma-Aldrich (Deisenhofen, Germany).
The cDNAs encoding for IFP and the three Arabidopsis thaliana proteins ANAC42, ARR1 and TPK1 were amplified by PCR using, respectively, the pENTR1A-IFP1.4&GFP vector , ANAC42 cDNA [AGI code: AT2G43000], the pDONR201-ARR1 vector , or TPK1 cDNA [AGI: AT5G55630] as templates. ANAC42 and ARR1 are transcription factors, whereas TPK1 is an ion channel. For TPK1, a partial cDNA encoding the N-terminal part of the channel protein (amino acids 1-79) was used . After amplification with PCR primers IFP sense, 5'-TCACCCATGGCTCGGGACCCTCTG-3' and IFP antisense, 5'-GTTGGTACCTTTATACAGCTCGTCCATTCC-3' IFP was cloned by restriction and ligation into the Nco I and Kpn I sites of the pLEXSY-sat2 vector (Jena Bioscience, Jena, Germany) encoding a C-terminal 6xHis epitope (fused to IFP) and a nourseothricin antibiotic resistance marker (Jena Bioscience). This vector, pLEXSY-IFP-His, was used to generate plasmids encoding C-terminally IFP-tagged ANAC42, ARR1 and TPK1 fusion proteins. To this end, ANAC42, ARR1 and TPK1 (1-79) sequences were PCR-amplified using the primers: ANAC42 sense, 5'-CACCCATGGGTGGCGAAGGTAACTTAGGTAAG-3', and ANAC42 antisense, 5'-TCACCCATGGCGGGTTTAGTGTTGCCATCTATAAC-3'; ARR1 sense, 5'-TCACCCATGGTGAATCCGAGTCACGGAAGAG-3', and ARR1 antisense, 5'-TCACCCATGGCAACCTGCTTAAGAAGTGCGCTC-3'; TPK1 sense, 5'-AAGAAGACATGTCGAGTGATGCAGCTC-3', and TPK1 antisense, 5'-AAGAAGACATGTCCACTCGCCTGAGATTCGG-3'. ANAC42- and ARR1-encoding PCR products were restricted and ligated into the Nco I site of vector pLEXSY-IFP-His. TPK1 (1-79)-encoding fragment was restricted by Pci I and ligated into the Nco I site of vector pLEXSY-IFP-His. The resulting constructs were named pLEXSY-ANAC42-IFP-His, pLEXSY-ARR1-IFP-His and pLEXSY-TPK1-IFP-His, respectively. The plasmids pLEXSY-IFP-ANAC42-His and pLEXSY-IFP-ARR1-His were generated in two steps for the expression of proteins N-terminally tagged with IFP. In the first step, ANAC42- and ARR1-encoding fragments were PCR amplified using the primers ANAC42 senseN, 5'-TCACCCATGGGTGGCGAAGGTAACTTAGGTAAG-3', and ANAC42 antisenseN 5'-TCCGCTAGCGGGTTTAGTGTTGCCATCTATAAC-3'; ARR1 senseN, 5'-TCACCCATGGTGAATCCGAGTCACGGAAGAG-3', and ARR1 antisenseN 5'-AAGAATGCTAGCAACCTGCTTAAGAAGTGCGCTC-3'. The resulting PCR products were then restricted and ligated into the Nco I and Nhe I sites of the pLEXSY-sat2 vector. These resulting constructs, pLEXSY-ANAC42-His and pLEXSY-ARR1-His, were then used in a second cloning step for the generation of the plasmids pLEXSY-IFP-ANAC42-His and pLEXSY-IFP-ARR1-His using the primers IFP sense and IFP antisense2, 5'-TCACCCATGGCTTTATACAGCTCGTCCATTCC-3', followed by restriction and ligation into the Nco I site of the vectors pLEXSY-ANAC42-His and pLEXSY-ARR1-His. pLEXSY-IFP-TPK1-His was generated using the primers TPK1 senseN, 5'-GTTGGTACCATGTCGAGTGATGCAGCTC-3' and TPK1 antisenseN, 5'-GTTGGTACCCACTCGCCTGAGATTCGG-3', followed by restriction and ligation into the Kpn I site of pLEXSY-IFP-His.
Protein expression and purification
IFP-6xHis, IFP-ANAC42-/ARR1-/TPK1-6xHis and ANAC42-/ARR1-/TPK1-IFP-6xHis fusion proteins were expressed using the LEXSYcon2 Expression Kit (Jena Bioscience). If not explicitly pointed out all components for cultivation of Leishmania cells and protein expression are included in the kit. Plasmids were transfected into Leishmania cells by electroporation and transfected cells were selected on nourseothricin-supplemented selective plates after five to seven days of incubation at 26°C according to the protocol included in the expression kit. Individual clones were selected and transferred sequentially each second day after incubation at 26°C into 96-well microtiter plates, from there into 24-well deep-well plates and than into 25 cm2 tissue culture flasks, filled with 150 μL, 1 mL and 10 mL selective medium. If not stated otherwise 96-well microtiter plates and 24-well deep-well plates were shaken at 60 rpm and tissue culture flasks were incubated in a static upright position. For protein stability and solubility tests 2 × 10 mL of an IFP expressing cell culture were pooled and centrifuged; cells were then sonicated in 1 mL standard Tris buffer in the absence or presence of protease inhibitors (1 mM EDTA, 1 mM PMSF, EDTA-free protease inhibitor cocktail; Roche, Mannheim, Germany). Supernatants of ultracentrifuged samples were analyzed. For protein purification, culture volume was scaled-up using nine 150 cm2-tissue culture flasks each containing 60 mL non-selective YE medium . After pooling all cultures, cells were centrifuged and the cell pellet was either stored at -80°C until use or immediately resuspended in standard Tris buffer supplemented with protease inhibitors (1 mM PMSF, EDTA-free protease inhibitor cocktail). Resuspended cells were sonicated and the supernatant of centrifuged samples was used for purification. IFP-His protein was purified using a 1-mL HisTrap HP column (GE Healthcare, Munich, Germany) coupled to an Äkta-Purifier FPLC system (GE Healthcare) and washing buffer supplemented with 40 mM imidazole. ANAC42-IFP-His was enriched using Protino Ni-IDA 150 packed column (Macherey-Nagel, Düren, Germany), in the presence of 1 mM EDTA to inhibit metal proteases, and washing buffer without imidazole.
Western blot and infrared analysis
Protein samples were separated in 12% SDS-polyacrylamide gels under denaturing conditions and analysed either (i) immunologically or by (ii) infrared scanning. (i) For immunological analysis SDS-PAGE-separated proteins were transferred onto Protran nitrocellulose membrane (Whatman, Kent, UK). The membrane was blocked for one hour in blocking buffer (5% non-fat dry milk in PBS containing 0.1% Tween-20), followed by incubation for 1 h with monoclonal mouse antibody (Santa Cruz Biotechnology, California, USA) directed against the 6xHis epitope. Membranes were washed three times for 10 min in wash buffer (PBS containing 0.1% Tween-20) and incubated for 1 h with IRDye800CW-conjugated goat anti-mouse secondary antibody (LI-COR, Bad Homburg, Germany). All incubations were performed at room temperature and antibodies were diluted 1:10,000 in blocking buffer. Signal intensities were analysed at 800 nm by using the Odyssey Infrared Imaging System (LI-COR). (ii) IFP-mediated infrared fluorescence was detected with the same system but scanning at 700 nm.
IFP was measured directly after transfer of IFP-His or ANAC42-IFP-His fusion protein expressing cells (2-100 μL) into the wells of black 96-well ELISA plates with clear flat bottom (Corning, New York, USA). If not stated otherwise, cell cultures were directly scanned in the wells of the ELISA plate; otherwise plates were centrifuged for 2 min at room temperature and 2500 rpm followed by scanning. In-gel detection was done after SDS-PAGE without demounting cast protein gels of the Mighty Small II system (Hoefer, Massachusetts, USA). Separated proteins were also visualized by Coomassie staining after infrared analysis.
For imaging of IFP in individual Leishmania cells fluorescence images were taken using a Zeiss Cell Observer HS/Axiovert 200 M deconvolution microscope (Carl Zeiss MicroImaging, Göttingen, Germany) equipped with a Cy5.5 filter set (665 ± 22.5 nm excitation and 725 ± 25 nm emission).
Results and Discussion
Expression of IFP in L. tarentolae
Expression levels of IFP and ANAC42, both carrying a 6xHis-tag at their C-terminal end, and their respective molecular weights were verified by SDS-PAGE and Western blot analysis (Figure 1B). Following the recommended procedural pipeline for protein expression in Leishmania (documented in the manual to the LEXSYcon2 Expression Kit; Jena Bioscience), we analyzed individual clones for accumulation of the heterologous proteins and identified four IFP-6xHis (no. 4, 5, 11 and 12) and three ANAC42-6xHis (no. 1, 2 and 3) clones expressing fusion protein (Figure 1B).
The role of hemin and biliverdin in IFP expressing Leishmania cells
IFP allows sensitive and rapid identification of well-expressing Leishmania clones
Online detection of IFP for the analysis of protein expression and purification parameters
In-gel detection of IFP and IFP fusion proteins
We demonstrated that IFP can be used as a facile reporter protein in L. tarentolae. Our study shows that IFP can be expressed in Leishmania cells to a level that allows easy detection by the Odyssey Infrared Imaging System, without compromising cell growth. Using IFP as biosensor expression of IFP and IFP fusion proteins in Leishmania is easily and rapidly detected in-cell, without cell disruption in microtiter plates. A few microliters of expression cultures are sufficient for in-cell detection and pre-selection of well-expressing Leishmania clones at an early stage of the protein production pipeline. Our IFP-based work-flow significantly reduces not only hands-on-time and work load for the individual researcher but also preserves precious cell culture, particularly when culture volumes and cell densities are low initially, when an expression protocol is established for a new protein, or when proteins are expressed weakly. Importantly, addition of biliverdin to the culture medium is not required to achieve infrared fluorescence of IFP-expressing Leishmania cells. Instead, hemin, a component essential for Leishmania growth, is sufficient for IFP chromophore formation, probably because of its intracellular enzymatic conversion to biliverdin.
As a further incentive, IFP fusion proteins retain infrared fluorescence after electrophoresis in denaturing SDS-polyacrylamide gels, allowing their direct in-gel detection by infrared imaging without disassembling cast gels. Thus, parameters for scaling up protein production and streamlining purification routes can be easily optimized when employing IFP as reporter. We envisage that expression of IFP-labelled proteins in Leishmania will assist the genomics-driven analysis or proteins from plants and other model organisms.
We gratefully acknowledge assistance from our technician (Karina Schulz) and a master student (Sandra Schmöckel), who contributed to the initial setup of the Leishmania expression system in the lab. We thank Ralph Gräph for introducing us to deconvolution microscopy. We thank Salma Balazadeh, Camilla Voelker and Alexander Heyl for providing cDNA templates for the proteins expressed here in Leishmania. We thank Roger Y. Tsien (Howard Hughes Medical Institute Laboratories, University of California, San Diego) for providing IFP coding fragment IFP1.4&GFP in pENTR1A. Funding was provided by the German Ministry of Education and Research (BMBF) within the InnoProfile initiative, project 'Integrated Protein Chips for the Point-of-Care Diagnostics - iPOC' (FKZ 03IP515).
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