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

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.

ORE1 balances leaf senescence against maintenance by antagonizing G2‐like‐mediated transcription

“…Primer sequences are given in supplementary Table S1 online. Expression of GST1–ORE1 and HIS–GLK2 fusion proteins in Escherichia coli strain Rosetta (DE3) pLysS (Merck), protein purification and in vitro pull‐down assays for immunological detection of purified GST–ORE1 and HIS–GLK2 fusion proteins were performed as described [17, 18]. For co‐immunoprecipitation of interacting protein complexes, protein was extracted from mesophyll cell protoplasts obtained from the VGE:ORE1::HA line transfected with the 35S:GFP or the 35S:GLK2::GFP construct.…”

NAC Transcription Factor SPEEDY HYPONASTIC GROWTH Regulates Flooding-Induced Leaf Movement in Arabidopsis

“…Proteins (15 µg) were separated on 12% polyacrylamide SDS gels and transferred to Protran nitrocellulose membrane (Whatman). Immunoblot analysis was performed as described (Dortay and Mueller-Roeber, 2010) using goat polyclonal IgG primary antibody directed against ACO (aE-18; Santa Cruz Biotechnology) and IRDye800CW donkey anti-goat IgG secondary antibody (LI-COR). Signal intensities were analyzed at 800 nm using the Odyssey Infrared Imaging System (LI-COR).…”

U-insertion/deletion RNA editing multiprotein complexes and mitochondrial ribosomes in Leishmania tarentolae are located in antipodal nodes adjacent to the kinetoplast DNA

“…These cells are non-pathogenic for mammals (Taylor et al, 2010) and grow readily in Brain Heart Infusion (BHI) (Difco) at 27˚C with hemin added to 10 μg/ml (Simpson et al, 1970). L. tarentolae has been studied extensively as a model trypanosomatid and has been recently developed and commercialized as a useful eukaryotic expression vector (Basile and Peticca, 2009; Breitling et al, 2002; Dortay and Mueller-Roeber, 2010; Fritsche et al, 2007; Klatt and Konthur, 2012; Kovtun et al, 2010; Kushnir et al, 2011; Kushnir et al, 2005). …”