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A highly efficient pipeline for protein expression in Leishmania tarentolae using infrared fluorescence protein as marker.

Dortay H, Mueller-Roeber B - Microb. Cell Fact. (2010)

Bottom Line: 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.

View Article: PubMed Central - HTML - PubMed

Affiliation: University of Potsdam, Institute of Biochemistry and Biology, Karl-Liebknecht-Strasse 24-25, Haus 20, Potsdam-Golm, Germany.

ABSTRACT

Background: 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.

Results: 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 microL - 100 microL). 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.

Conclusions: 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.

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Identification of well-expressing Leishmania strains. A) Four IFP-His and three ANAC42-His fusion protein-expressing Leishmania cell lines (clones 4, 5, 11, 12 and 1, 2, 3, respectively) of different volumes (2 μL, 10 μL and 100 μL) were transferred into the wells of a 96-well ELISA plate and centrifuged for 2 min at 2,500 g, followed by pipeting again 100 μL of the respective cell lines into neighbouring wells indicated by a white-dashed box. IR scanning was performed using the Odyssey Infrared Imaging System. B) Schematic outline of the previously established LEXSY protocol and the IFP protocol described here. The flow-chart high-lights the differences of the two Leishmania protein expression protocols in terms of expenditures of time and effort. C) Twelve different IFP fusion protein (1, ARR1-IFP-His; 2, IFP-ARR1-His; 3, ANAC42-IFP-His; 4, IFP-ANAC42-His; 5, TPK1-IFP-His; 6, IFP-TPK1-His) expressing Leishmania cell lines were picked from the selective plate and transferred into the wells of a 96-well microtiter plate filled with 150 μL BHI medium, followed by two days of incubation and IR scan (upper panel). The complete volumes of all cell lines were transferred into the wells of a 24-well deep-well plate containing 1 mL BHI medium, followed by incubation for two days. 100 μL-samples of each cell line were IR-scanned in the wells of a 96-well microtiter plate (lower panel). IFP-His expressing cell line was used as positive control (P) and ANAC42-His expressing cell line as negative control (N).
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Figure 3: Identification of well-expressing Leishmania strains. A) Four IFP-His and three ANAC42-His fusion protein-expressing Leishmania cell lines (clones 4, 5, 11, 12 and 1, 2, 3, respectively) of different volumes (2 μL, 10 μL and 100 μL) were transferred into the wells of a 96-well ELISA plate and centrifuged for 2 min at 2,500 g, followed by pipeting again 100 μL of the respective cell lines into neighbouring wells indicated by a white-dashed box. IR scanning was performed using the Odyssey Infrared Imaging System. B) Schematic outline of the previously established LEXSY protocol and the IFP protocol described here. The flow-chart high-lights the differences of the two Leishmania protein expression protocols in terms of expenditures of time and effort. C) Twelve different IFP fusion protein (1, ARR1-IFP-His; 2, IFP-ARR1-His; 3, ANAC42-IFP-His; 4, IFP-ANAC42-His; 5, TPK1-IFP-His; 6, IFP-TPK1-His) expressing Leishmania cell lines were picked from the selective plate and transferred into the wells of a 96-well microtiter plate filled with 150 μL BHI medium, followed by two days of incubation and IR scan (upper panel). The complete volumes of all cell lines were transferred into the wells of a 24-well deep-well plate containing 1 mL BHI medium, followed by incubation for two days. 100 μL-samples of each cell line were IR-scanned in the wells of a 96-well microtiter plate (lower panel). IFP-His expressing cell line was used as positive control (P) and ANAC42-His expressing cell line as negative control (N).

Mentions: In order to demonstrate the sensitivity of IFP as an in vivo detector for protein synthesis different IFP expressing cell lines were grown in 25 cm2 tissue culture flasks in a culture volume of 10 mL, as recommended by the manufacturer. Subsequently 2 μL, 10 μL and 100 μL, respectively, of the cell culture were transferred into individual wells of a 96-well ELISA plate with a flat and clear bottom, and centrifuged. After centrifugation a second transfer of 100 μL culture volume into neighboring wells was carried out. Centrifuged and non-centrifuged IFP expressing Leishmania cell cultures were scanned directly in the ELISA plate. Cell lines expressing ANAC42 protein without a fusion to IFP were used as negative control. As can be seen in Figure 3A, culture volumes of as little as 2 μL are sufficient for straightforward detection of IFP expressing Leishmania; furthermore centrifugation to collect cells in a pellet is not required for in vivo IFP detection (dashed white box in Figure 3A). Thus, IFP fusion proteins are rapidly detected by IR measurement allowing pre-selection of well-expressing Leishmania cell lines at an early stage of the expression pipeline. In Figure 3B we compare the Leishmania protein expression work-flow (LEXSY protocol) recommended by the manufacturer, which uses 6xHis as reporter, with the work-flow reported here that employs IFP as marker (IFP protocol). According to the IFP protocol well-expressing Leishmania lines are visualized by IR scan in a 96-well microtiter plate already two days after transfer and incubation of individual clones in a culture volume of 150 μL. The complete culture volumes of each clone are then transferred into the wells of a 24-well deep-well plate (containing 1 mL culture broth each), followed by two additional days of incubation. IR scan in a 96-well microtiter plate, using 100 μL of each clone, enables a pre-selection of cell lines expressing IFP at moderate to high level. Subsequently, 1 mL culture of well-expressing clones is transferred into 10 mL culture broth in 25 cm2 tissue culture flasks. After two further days of incubation, at day six, IFP or IFP fusion proteins are detected in-gel after separation by SDS-PAGE (IFP protocol in Figure 3B). In contrast, in the LEXSY protocol all cultures are scaled-up for the detection of 6xHis fusion proteins by Western blot analysis after seven to eight days in total. Thus, time (1 1/2 days) and in particular experimental effort required for the detection of protein expression in Leishmania can be reduced considerably when using IFP as reporter (Figure 3). The new protocol was approved by expressing different proteins fused to IFP at their N- and C-terminal ends, respectively, including ARR1, a response regulator in the plant cytokinin signaling pathway, ANAC42, a transcription factor, and the N-terminal GRF (General Regulating Factor)-interacting domain of TPK1 (Figure 3C). Ten μL each of 12 randomly selected cell lines of unknown cell densities were centrifuged and analyzed for IR fluorescence at two different stages of the newly established protein expression work-flow, namely two days after incubation in the 96-well microtiter plate (Figure 3C, upper panel) and two days after incubation in the 24-well deep-well plate (Figure 3C, lower panel). Taken together, the utilization of IFP as a reporter for expression of fusion proteins in Leishmania reduces time, costs and efforts and thus makes it an attractive reporter protein especially for high-throughput analyses in genomics research.


A highly efficient pipeline for protein expression in Leishmania tarentolae using infrared fluorescence protein as marker.

Dortay H, Mueller-Roeber B - Microb. Cell Fact. (2010)

Identification of well-expressing Leishmania strains. A) Four IFP-His and three ANAC42-His fusion protein-expressing Leishmania cell lines (clones 4, 5, 11, 12 and 1, 2, 3, respectively) of different volumes (2 μL, 10 μL and 100 μL) were transferred into the wells of a 96-well ELISA plate and centrifuged for 2 min at 2,500 g, followed by pipeting again 100 μL of the respective cell lines into neighbouring wells indicated by a white-dashed box. IR scanning was performed using the Odyssey Infrared Imaging System. B) Schematic outline of the previously established LEXSY protocol and the IFP protocol described here. The flow-chart high-lights the differences of the two Leishmania protein expression protocols in terms of expenditures of time and effort. C) Twelve different IFP fusion protein (1, ARR1-IFP-His; 2, IFP-ARR1-His; 3, ANAC42-IFP-His; 4, IFP-ANAC42-His; 5, TPK1-IFP-His; 6, IFP-TPK1-His) expressing Leishmania cell lines were picked from the selective plate and transferred into the wells of a 96-well microtiter plate filled with 150 μL BHI medium, followed by two days of incubation and IR scan (upper panel). The complete volumes of all cell lines were transferred into the wells of a 24-well deep-well plate containing 1 mL BHI medium, followed by incubation for two days. 100 μL-samples of each cell line were IR-scanned in the wells of a 96-well microtiter plate (lower panel). IFP-His expressing cell line was used as positive control (P) and ANAC42-His expressing cell line as negative control (N).
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Figure 3: Identification of well-expressing Leishmania strains. A) Four IFP-His and three ANAC42-His fusion protein-expressing Leishmania cell lines (clones 4, 5, 11, 12 and 1, 2, 3, respectively) of different volumes (2 μL, 10 μL and 100 μL) were transferred into the wells of a 96-well ELISA plate and centrifuged for 2 min at 2,500 g, followed by pipeting again 100 μL of the respective cell lines into neighbouring wells indicated by a white-dashed box. IR scanning was performed using the Odyssey Infrared Imaging System. B) Schematic outline of the previously established LEXSY protocol and the IFP protocol described here. The flow-chart high-lights the differences of the two Leishmania protein expression protocols in terms of expenditures of time and effort. C) Twelve different IFP fusion protein (1, ARR1-IFP-His; 2, IFP-ARR1-His; 3, ANAC42-IFP-His; 4, IFP-ANAC42-His; 5, TPK1-IFP-His; 6, IFP-TPK1-His) expressing Leishmania cell lines were picked from the selective plate and transferred into the wells of a 96-well microtiter plate filled with 150 μL BHI medium, followed by two days of incubation and IR scan (upper panel). The complete volumes of all cell lines were transferred into the wells of a 24-well deep-well plate containing 1 mL BHI medium, followed by incubation for two days. 100 μL-samples of each cell line were IR-scanned in the wells of a 96-well microtiter plate (lower panel). IFP-His expressing cell line was used as positive control (P) and ANAC42-His expressing cell line as negative control (N).
Mentions: In order to demonstrate the sensitivity of IFP as an in vivo detector for protein synthesis different IFP expressing cell lines were grown in 25 cm2 tissue culture flasks in a culture volume of 10 mL, as recommended by the manufacturer. Subsequently 2 μL, 10 μL and 100 μL, respectively, of the cell culture were transferred into individual wells of a 96-well ELISA plate with a flat and clear bottom, and centrifuged. After centrifugation a second transfer of 100 μL culture volume into neighboring wells was carried out. Centrifuged and non-centrifuged IFP expressing Leishmania cell cultures were scanned directly in the ELISA plate. Cell lines expressing ANAC42 protein without a fusion to IFP were used as negative control. As can be seen in Figure 3A, culture volumes of as little as 2 μL are sufficient for straightforward detection of IFP expressing Leishmania; furthermore centrifugation to collect cells in a pellet is not required for in vivo IFP detection (dashed white box in Figure 3A). Thus, IFP fusion proteins are rapidly detected by IR measurement allowing pre-selection of well-expressing Leishmania cell lines at an early stage of the expression pipeline. In Figure 3B we compare the Leishmania protein expression work-flow (LEXSY protocol) recommended by the manufacturer, which uses 6xHis as reporter, with the work-flow reported here that employs IFP as marker (IFP protocol). According to the IFP protocol well-expressing Leishmania lines are visualized by IR scan in a 96-well microtiter plate already two days after transfer and incubation of individual clones in a culture volume of 150 μL. The complete culture volumes of each clone are then transferred into the wells of a 24-well deep-well plate (containing 1 mL culture broth each), followed by two additional days of incubation. IR scan in a 96-well microtiter plate, using 100 μL of each clone, enables a pre-selection of cell lines expressing IFP at moderate to high level. Subsequently, 1 mL culture of well-expressing clones is transferred into 10 mL culture broth in 25 cm2 tissue culture flasks. After two further days of incubation, at day six, IFP or IFP fusion proteins are detected in-gel after separation by SDS-PAGE (IFP protocol in Figure 3B). In contrast, in the LEXSY protocol all cultures are scaled-up for the detection of 6xHis fusion proteins by Western blot analysis after seven to eight days in total. Thus, time (1 1/2 days) and in particular experimental effort required for the detection of protein expression in Leishmania can be reduced considerably when using IFP as reporter (Figure 3). The new protocol was approved by expressing different proteins fused to IFP at their N- and C-terminal ends, respectively, including ARR1, a response regulator in the plant cytokinin signaling pathway, ANAC42, a transcription factor, and the N-terminal GRF (General Regulating Factor)-interacting domain of TPK1 (Figure 3C). Ten μL each of 12 randomly selected cell lines of unknown cell densities were centrifuged and analyzed for IR fluorescence at two different stages of the newly established protein expression work-flow, namely two days after incubation in the 96-well microtiter plate (Figure 3C, upper panel) and two days after incubation in the 24-well deep-well plate (Figure 3C, lower panel). Taken together, the utilization of IFP as a reporter for expression of fusion proteins in Leishmania reduces time, costs and efforts and thus makes it an attractive reporter protein especially for high-throughput analyses in genomics research.

Bottom Line: 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.

View Article: PubMed Central - HTML - PubMed

Affiliation: University of Potsdam, Institute of Biochemistry and Biology, Karl-Liebknecht-Strasse 24-25, Haus 20, Potsdam-Golm, Germany.

ABSTRACT

Background: 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.

Results: 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 microL - 100 microL). 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.

Conclusions: 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.

Show MeSH