Abstract
BackgroundLeishmania 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.ResultsIFP 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.ConclusionsUsing 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.
Highlights
Leishmania tarentolae, a unicellular eukaryotic protozoan, has been established as a novel host for recombinant protein production in recent years
Since its introduction as a new host for protein production which benefited from the development of methods for trypanosomatid cultivation and their genetic manipulation [6], Leishmania tarentolae has been used for the successful expression of various heterologous proteins such as e.g. proprotein convertase 4
In order to investigate the potential usage of infrared fluorescence protein (IFP) as a reporter for protein expression in L. tarentolae we expressed it in the cytoplasm and analyzed the cells by deconvolution microscopy (Figure 1A)
Summary
Leishmania tarentolae, a unicellular eukaryotic protozoan, has been established as a novel host for recombinant protein production in recent years. 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 [1]. Since its introduction as a new host for protein production which benefited from the development of methods for trypanosomatid cultivation and their genetic manipulation [6], Leishmania tarentolae has been used for the successful expression of various heterologous proteins such as e.g. proprotein convertase 4 It was 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 [10]. Successful expression of plant proteins in Leishmania cells or in vitro extracts has to our knowledge not been reported so far
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