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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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Online detection of IFP in L. tarentolae. IFP-His expressing Leishmania cells were grown in 25 cm2-tissue culture flasks in 10 mL of BHI medium in static upright (SU), static flat (SF) and dynamic (60 rpm) flat (A) position. ANAC42-His expressing cells were used as negative control. After 0, 6, 24, 48, 72 and 96 h of incubation 100 μL-samples were IR-scanned in the wells of a 96-well microtiter plate.
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Figure 4: Online detection of IFP in L. tarentolae. IFP-His expressing Leishmania cells were grown in 25 cm2-tissue culture flasks in 10 mL of BHI medium in static upright (SU), static flat (SF) and dynamic (60 rpm) flat (A) position. ANAC42-His expressing cells were used as negative control. After 0, 6, 24, 48, 72 and 96 h of incubation 100 μL-samples were IR-scanned in the wells of a 96-well microtiter plate.

Mentions: Production of large quantities of proteins with high yield requires optimal expression parameters. Once expression is optimized under laboratory conditions, the process can be scaled-up to the desired bioreactor volume followed by purification of the protein of interest. Besides optimizing gene sequences and choosing the right expression system, optimal expression yields can be achieved by changing culture and bioreactor parameters, e.g. media ingredients, pH value or O2 concentration in the culture broth. To demonstrate that IFP can be used as an online sensor allowing researchers to continuously and quantitatively monitor expression of IFP in real time, a 1:10 diluted IFP-expressing Leishmania cell culture was grown in 25 cm2 tissue culture flasks in a volume of 10 mL under different mechanical conditions (static upright, static flat and dynamic flat). Changes in IR signal intensities due to changes of IFP quantities were measured at different time points by transferring and scanning 100 μL of the cell cultures directly into the wells of a 96-well ELISA plate without centrifugation (Figure 4). After 24 h of incubation the first IFP signal among all three incubation conditions was detected and a further increase of the signal was observed after two, three and four days, respectively. However, dynamic flat incubation resulted in only a slow increase of the IFP signal, while signal intensity increased rapidly when cells were incubated under static upright condition, and even faster when grown under static flat condition. In the latter case, high level of IFP was observed after 72 h and 96 h, visible as white pixels in the image (Figure 4). These results indicate that IFP expression under dynamic incubation condition can inhibit the production of IFP due to either excessive oxygen transfer into the culture broth or increased shear forces acting on the Leishmania cells due to movement of the culture flasks. Next we tested the stability and solubility of IFP in two different standard buffers used for the purification of His-tagged fusion proteins, in the absence and presence of different protease inhibitors. Figure 5 demonstrates that IFP alone is stable in Tris and PBS buffer in the absence of protease inhibitors and accumulates in the soluble supernatant after ultracentrifugation, which is a prerequisite for purification of proteins under native conditions. Cells were disrupted in the described buffers by ultrasound treatment indicating that sonication does not affect the infrared signal of IFP. IFP production was scaled-up by growing cells in parallel in nine 150 cm2 tissue culture flasks in a culture volume of 60 mL. The flasks were incubated static flat for four days, and the development of IR signal in each flask was monitored every 24 h (Figure 6A). All culture broths were pooled and centrifuged on the fourth day. The resulting cell pellet was disrupted in 25 mL Tris buffer with an EDTA-free protease inhibitor cocktail and IFP-His fusion protein was purified by Äkta-FPLC using a 1-mL His-Trap column. Unbound flow-through, as well as flow-through of washing and elution steps were collected (1-mL fractions). Hundred μl of each fraction were analyzed by IR scan allowing the detection of bound and partially unbound IFP-His fusion protein (Figure 6B). The amount of unbound protein can be reduced by collecting and recirculating the unbound flow through several times through the column. This was demonstrated by using the ANAC42-IFP-His fusion protein and recirculating it three times manually through a gravity flow-based 6xHis-affinity column (Figure 6C).


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)

Online detection of IFP in L. tarentolae. IFP-His expressing Leishmania cells were grown in 25 cm2-tissue culture flasks in 10 mL of BHI medium in static upright (SU), static flat (SF) and dynamic (60 rpm) flat (A) position. ANAC42-His expressing cells were used as negative control. After 0, 6, 24, 48, 72 and 96 h of incubation 100 μL-samples were IR-scanned in the wells of a 96-well microtiter plate.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2874516&req=5

Figure 4: Online detection of IFP in L. tarentolae. IFP-His expressing Leishmania cells were grown in 25 cm2-tissue culture flasks in 10 mL of BHI medium in static upright (SU), static flat (SF) and dynamic (60 rpm) flat (A) position. ANAC42-His expressing cells were used as negative control. After 0, 6, 24, 48, 72 and 96 h of incubation 100 μL-samples were IR-scanned in the wells of a 96-well microtiter plate.
Mentions: Production of large quantities of proteins with high yield requires optimal expression parameters. Once expression is optimized under laboratory conditions, the process can be scaled-up to the desired bioreactor volume followed by purification of the protein of interest. Besides optimizing gene sequences and choosing the right expression system, optimal expression yields can be achieved by changing culture and bioreactor parameters, e.g. media ingredients, pH value or O2 concentration in the culture broth. To demonstrate that IFP can be used as an online sensor allowing researchers to continuously and quantitatively monitor expression of IFP in real time, a 1:10 diluted IFP-expressing Leishmania cell culture was grown in 25 cm2 tissue culture flasks in a volume of 10 mL under different mechanical conditions (static upright, static flat and dynamic flat). Changes in IR signal intensities due to changes of IFP quantities were measured at different time points by transferring and scanning 100 μL of the cell cultures directly into the wells of a 96-well ELISA plate without centrifugation (Figure 4). After 24 h of incubation the first IFP signal among all three incubation conditions was detected and a further increase of the signal was observed after two, three and four days, respectively. However, dynamic flat incubation resulted in only a slow increase of the IFP signal, while signal intensity increased rapidly when cells were incubated under static upright condition, and even faster when grown under static flat condition. In the latter case, high level of IFP was observed after 72 h and 96 h, visible as white pixels in the image (Figure 4). These results indicate that IFP expression under dynamic incubation condition can inhibit the production of IFP due to either excessive oxygen transfer into the culture broth or increased shear forces acting on the Leishmania cells due to movement of the culture flasks. Next we tested the stability and solubility of IFP in two different standard buffers used for the purification of His-tagged fusion proteins, in the absence and presence of different protease inhibitors. Figure 5 demonstrates that IFP alone is stable in Tris and PBS buffer in the absence of protease inhibitors and accumulates in the soluble supernatant after ultracentrifugation, which is a prerequisite for purification of proteins under native conditions. Cells were disrupted in the described buffers by ultrasound treatment indicating that sonication does not affect the infrared signal of IFP. IFP production was scaled-up by growing cells in parallel in nine 150 cm2 tissue culture flasks in a culture volume of 60 mL. The flasks were incubated static flat for four days, and the development of IR signal in each flask was monitored every 24 h (Figure 6A). All culture broths were pooled and centrifuged on the fourth day. The resulting cell pellet was disrupted in 25 mL Tris buffer with an EDTA-free protease inhibitor cocktail and IFP-His fusion protein was purified by Äkta-FPLC using a 1-mL His-Trap column. Unbound flow-through, as well as flow-through of washing and elution steps were collected (1-mL fractions). Hundred μl of each fraction were analyzed by IR scan allowing the detection of bound and partially unbound IFP-His fusion protein (Figure 6B). The amount of unbound protein can be reduced by collecting and recirculating the unbound flow through several times through the column. This was demonstrated by using the ANAC42-IFP-His fusion protein and recirculating it three times manually through a gravity flow-based 6xHis-affinity column (Figure 6C).

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