l tarentolae parrot tar  (ATCC)

Journal: bioRxiv

Article Title: Novel Binding Partners of the Vacuolar Transporter Chaperone (VTC) complex in Acidocalcisomes of Leishmania tarentolae

doi: 10.1101/2025.09.23.677757

Figure Lengend Snippet: CRISPR/Cas9 tagging and expression of LtVtc1 and LtVtc4. (A) Schematic representation of LtVtc1 and LtVtc4. SPX: SYG1/Pho81/XPR domain, CD: Catalytic domain, TMH: Transmembrane helix domain. (B) PCR verification of mNG-tagged LtVtc1 and LtVtc4 showing a band at ∼2200 bp, absent in WT. (C) Fluorescent detection of mNG-LtLtVtc1, and LtVtc4 expressed in L. tarentolae ; Coomassie blue staining of WT L. tarentolae , mNG-LtVtc1, mNG-LtVtc4 confirms comparable total protein levels, ensuring consistent sample loading for fluorescent detection. (D) Fluorescence microscopy micrographs of mNG-LtLtVtc1 and mNG-LtVtc4 with mPPase in L.tarentolae (Pearson’s r = 0.85 and 0.83, respectively), scale bar: 5 µm.

Article Snippet: GO analysis of biological processes, cell components, and molecular functions was performed on the 412 overlapping LtVtc1 and LtVtc4 interactors using the TriTrypDB GO analysis tool [ ] with the complete L. tarentolae parrot tar II 2019 (ATCC 30267) genome as the reference.

Techniques: CRISPR, Expressing, Staining, Fluorescence, Microscopy

Journal: bioRxiv

Article Title: Novel Binding Partners of the Vacuolar Transporter Chaperone (VTC) complex in Acidocalcisomes of Leishmania tarentolae

doi: 10.1101/2025.09.23.677757

Figure Lengend Snippet: Colocalisation analysis of L. tarentolae expressing mNG-LtVtc4 and mCh-tagged by immunofluorescence microscopy. mNG-LtVtc4 colocalises with mCh-LtVtc1 (Pearson’s r = 0.83) as well as with mCh-LtPho91, mCh-LtCa²⁺-ATPase, mCh-LtV-H⁺-ATPase_A, and mCh-LtZnT, showing Pearson’s correlation coefficients of 0.87, 0.83, 0.87, and 0.84, respectively. A lower degree of colocalisation is observed with mCh-IP₃R (r = 0.67). Scale bar: 5 µm.

Article Snippet: GO analysis of biological processes, cell components, and molecular functions was performed on the 412 overlapping LtVtc1 and LtVtc4 interactors using the TriTrypDB GO analysis tool [ ] with the complete L. tarentolae parrot tar II 2019 (ATCC 30267) genome as the reference.

Techniques: Expressing, Immunofluorescence, Microscopy

Journal: bioRxiv

Article Title: Novel Binding Partners of the Vacuolar Transporter Chaperone (VTC) complex in Acidocalcisomes of Leishmania tarentolae

doi: 10.1101/2025.09.23.677757

Figure Lengend Snippet:

Article Snippet: GO analysis of biological processes, cell components, and molecular functions was performed on the 412 overlapping LtVtc1 and LtVtc4 interactors using the TriTrypDB GO analysis tool [ ] with the complete L. tarentolae parrot tar II 2019 (ATCC 30267) genome as the reference.

Techniques: Binding Assay

Journal: bioRxiv

Article Title: Novel Binding Partners of the Vacuolar Transporter Chaperone (VTC) complex in Acidocalcisomes of Leishmania tarentolae

doi: 10.1101/2025.09.23.677757

Figure Lengend Snippet: Colocalisation analysis of L. tarentolae expressing mNG-LtVtc4 and mCh-tagged LtVBP1, LtVBP2, and LtVBP3 by immunofluorescence microscopy. All three proteins show colocalisation with LtVtc4 in acidocalcisomes. Pearson’s correlation coefficients with LtVtc4 are 0.83 for LtVBP1, 0.85 for LtVBP2, and 0.82 for LtVBP3. Scale bar: 5 µm.

Article Snippet: GO analysis of biological processes, cell components, and molecular functions was performed on the 412 overlapping LtVtc1 and LtVtc4 interactors using the TriTrypDB GO analysis tool [ ] with the complete L. tarentolae parrot tar II 2019 (ATCC 30267) genome as the reference.

Techniques: Expressing, Immunofluorescence, Microscopy

Journal: bioRxiv

Article Title: Novel Binding Partners of the Vacuolar Transporter Chaperone (VTC) complex in Acidocalcisomes of Leishmania tarentolae

doi: 10.1101/2025.09.23.677757

Figure Lengend Snippet: Pulldown assay of novel VTC binding partners with mNG-LtVtc4 in L. tarentolae. (A-C) SDS-PAGE with fluorescence detection shows capture of mCh-LtVBP1, mCh-LtVBP2, and mCh-LtVBP3 by mNG-LtVtc4 using mNeonGreen-Trap agarose beads, as indicated by signal in the bound fraction. (D) Pulldown of mNG-LtVtc4 with mCh-LtVBP3 using ChromoTek RFP-Trap beads confirms interaction. Lane labels: I – input; FT – flow-through; W3 – third wash; B – bound fraction. Green bands correspond to mNG-LtVtc4; some I and B lines appear as yellowish due to partial detection of the mNG tag in the 532 nm channel ( S6 Fig ). Red bands correspond to mCherry-tagged proteins. The fluorescence signal marked by a white asterisk in the gel represents free mCh tag, visible only in the input due to partial degradation.

Article Snippet: GO analysis of biological processes, cell components, and molecular functions was performed on the 412 overlapping LtVtc1 and LtVtc4 interactors using the TriTrypDB GO analysis tool [ ] with the complete L. tarentolae parrot tar II 2019 (ATCC 30267) genome as the reference.

Techniques: Binding Assay, SDS Page, Fluorescence

Journal: bioRxiv

Article Title: Novel Binding Partners of the Vacuolar Transporter Chaperone (VTC) complex in Acidocalcisomes of Leishmania tarentolae

doi: 10.1101/2025.09.23.677757

Figure Lengend Snippet: CRISPR/Cas9 tagging and expression of LtVtc1 and LtVtc4. (A) Schematic representation of LtVtc1 and LtVtc4. SPX: SYG1/Pho81/XPR domain, CD: Catalytic domain, TMH: Transmembrane helix domain. (B) PCR verification of mNG-tagged LtVtc1 and LtVtc4 showing a band at ∼2200 bp, absent in WT. (C) Fluorescent detection of mNG-LtLtVtc1, and LtVtc4 expressed in L. tarentolae ; Coomassie blue staining of WT L. tarentolae , mNG-LtVtc1, mNG-LtVtc4 confirms comparable total protein levels, ensuring consistent sample loading for fluorescent detection. (D) Fluorescence microscopy micrographs of mNG-LtLtVtc1 and mNG-LtVtc4 with mPPase in L.tarentolae (Pearson’s r = 0.85 and 0.83, respectively), scale bar: 5 µm.

Article Snippet: L. tarentolae P10 strain promastigotes (LEXSY host P10, Jena Biosciences) were cultured at 27 °C in ventilated tissue culture flasks in 10 ml of brain heart infusion (BHI) medium containing 37 mg/mL BHI powder (Lexsy Broth BHI, Jena Biosciences) supplemented with 5 μg/mL hemin as described by the manufacturer (Jena Biosciences).

Techniques: CRISPR, Expressing, Staining, Fluorescence, Microscopy

Journal: bioRxiv

Article Title: Novel Binding Partners of the Vacuolar Transporter Chaperone (VTC) complex in Acidocalcisomes of Leishmania tarentolae

doi: 10.1101/2025.09.23.677757

Figure Lengend Snippet: Colocalisation analysis of L. tarentolae expressing mNG-LtVtc4 and mCh-tagged by immunofluorescence microscopy. mNG-LtVtc4 colocalises with mCh-LtVtc1 (Pearson’s r = 0.83) as well as with mCh-LtPho91, mCh-LtCa²⁺-ATPase, mCh-LtV-H⁺-ATPase_A, and mCh-LtZnT, showing Pearson’s correlation coefficients of 0.87, 0.83, 0.87, and 0.84, respectively. A lower degree of colocalisation is observed with mCh-IP₃R (r = 0.67). Scale bar: 5 µm.

Article Snippet: L. tarentolae P10 strain promastigotes (LEXSY host P10, Jena Biosciences) were cultured at 27 °C in ventilated tissue culture flasks in 10 ml of brain heart infusion (BHI) medium containing 37 mg/mL BHI powder (Lexsy Broth BHI, Jena Biosciences) supplemented with 5 μg/mL hemin as described by the manufacturer (Jena Biosciences).

Techniques: Expressing, Immunofluorescence, Microscopy

Journal: bioRxiv

Article Title: Novel Binding Partners of the Vacuolar Transporter Chaperone (VTC) complex in Acidocalcisomes of Leishmania tarentolae

doi: 10.1101/2025.09.23.677757

Figure Lengend Snippet:

Article Snippet: L. tarentolae P10 strain promastigotes (LEXSY host P10, Jena Biosciences) were cultured at 27 °C in ventilated tissue culture flasks in 10 ml of brain heart infusion (BHI) medium containing 37 mg/mL BHI powder (Lexsy Broth BHI, Jena Biosciences) supplemented with 5 μg/mL hemin as described by the manufacturer (Jena Biosciences).

Techniques: Binding Assay

Journal: bioRxiv

Article Title: Novel Binding Partners of the Vacuolar Transporter Chaperone (VTC) complex in Acidocalcisomes of Leishmania tarentolae

doi: 10.1101/2025.09.23.677757

Figure Lengend Snippet: Colocalisation analysis of L. tarentolae expressing mNG-LtVtc4 and mCh-tagged LtVBP1, LtVBP2, and LtVBP3 by immunofluorescence microscopy. All three proteins show colocalisation with LtVtc4 in acidocalcisomes. Pearson’s correlation coefficients with LtVtc4 are 0.83 for LtVBP1, 0.85 for LtVBP2, and 0.82 for LtVBP3. Scale bar: 5 µm.

Article Snippet: L. tarentolae P10 strain promastigotes (LEXSY host P10, Jena Biosciences) were cultured at 27 °C in ventilated tissue culture flasks in 10 ml of brain heart infusion (BHI) medium containing 37 mg/mL BHI powder (Lexsy Broth BHI, Jena Biosciences) supplemented with 5 μg/mL hemin as described by the manufacturer (Jena Biosciences).

Techniques: Expressing, Immunofluorescence, Microscopy

Journal: bioRxiv

Article Title: Novel Binding Partners of the Vacuolar Transporter Chaperone (VTC) complex in Acidocalcisomes of Leishmania tarentolae

doi: 10.1101/2025.09.23.677757

Figure Lengend Snippet: Pulldown assay of novel VTC binding partners with mNG-LtVtc4 in L. tarentolae. (A-C) SDS-PAGE with fluorescence detection shows capture of mCh-LtVBP1, mCh-LtVBP2, and mCh-LtVBP3 by mNG-LtVtc4 using mNeonGreen-Trap agarose beads, as indicated by signal in the bound fraction. (D) Pulldown of mNG-LtVtc4 with mCh-LtVBP3 using ChromoTek RFP-Trap beads confirms interaction. Lane labels: I – input; FT – flow-through; W3 – third wash; B – bound fraction. Green bands correspond to mNG-LtVtc4; some I and B lines appear as yellowish due to partial detection of the mNG tag in the 532 nm channel ( S6 Fig ). Red bands correspond to mCherry-tagged proteins. The fluorescence signal marked by a white asterisk in the gel represents free mCh tag, visible only in the input due to partial degradation.

Article Snippet: L. tarentolae P10 strain promastigotes (LEXSY host P10, Jena Biosciences) were cultured at 27 °C in ventilated tissue culture flasks in 10 ml of brain heart infusion (BHI) medium containing 37 mg/mL BHI powder (Lexsy Broth BHI, Jena Biosciences) supplemented with 5 μg/mL hemin as described by the manufacturer (Jena Biosciences).

Techniques: Binding Assay, SDS Page, Fluorescence

Journal: bioRxiv

Article Title: A conserved mechanism for the retrieval of polyubiquitinated proteins from cilia

doi: 10.1101/2025.04.24.650332

Figure Lengend Snippet: (A) Schematic workflow of pulldown experiment from flagellar lysates using immobilized K48- and K63-linked ubiquitin chains and subsequent quantitative multiplexed mass spectrometry through tandem mass tagging (TMT-MS) analysis. (B, C) Heatmaps showing quantification of significantly enriched ubiquitin readers in C. reinhardtii (B) and L. tarentolae (C) flagellar lysates identified in triplicate or quadruplicate pulldowns (R1-R4). A one-way ANOVA was performed for each protein across experimental groups, followed by Ben-jamini-Hochberg false discovery rate (FDR) correction for multiple testing. Proteins with q-values below 0.01 were considered statistically significant, and those exhibiting a fold change greater than 2 compared to the empty beads control group were retained. (D) Venn diagram of proteins identified in the TMT-MS analysis of C. reinhardtii and L. tarentolae ubiquitin pulldowns. Only homologs of CFAP36, TOM1L2 and USP5 were identified in pulldowns from both organisms.

Article Snippet: Briefly, L. tarentolae strain P10 (Jena Bioscience, #LT-101) were grown in BHI medium (HIMEDIA, #N210) supplemented with 5 mg/mL hemin chloride (Sigma, #3741) and 10 U/mL Penicillin-Streptomycin (Gibco, #15070063) at 26 °C.

Techniques: Mass Spectrometry, Control

Journal: bioRxiv

Article Title: A conserved mechanism for the retrieval of polyubiquitinated proteins from cilia

doi: 10.1101/2025.04.24.650332

Figure Lengend Snippet: Related to . (A)SDS-PAGE analysis of biotinylated K48- and K63-linked polyubiquitin (pUb) chains before and after addition of streptavidin (SA) beads. (B-C) SDS-PAGE analysis of elution from empty and K48- or K63-linked ubiquitin-coated beads incubated with flagellar extracts from C. reinhardtii (B)and L. tarentolae (C). Each elution was analyzed by quantitative mass spectrometry. Replicate number is shown above each lane. (D)Volcano plots generated from TMT-MS analysis of flagellar extracts from C. reinhardtii comparing protein binding to empty beads versus beads coated with K48-linked (left) or K63-linked (right) polyubiquitin chains. Each plot shows significance versus enrichment. Individual proteins are shown as gray dots except for ARL3, CFAP36, TOM1L2, and USP5, which are colored and labeled. (E)Volcano plots generated from TMT-MS analysis of flagellar extracts from L. tarentolae comparing protein binding to empty beads versus beads coated with K48-linked (left) or K63-linked (right) ubiquitin chains in the absence (top) or presence (bottom) of GTP.

Article Snippet: Briefly, L. tarentolae strain P10 (Jena Bioscience, #LT-101) were grown in BHI medium (HIMEDIA, #N210) supplemented with 5 mg/mL hemin chloride (Sigma, #3741) and 10 U/mL Penicillin-Streptomycin (Gibco, #15070063) at 26 °C.

Techniques: SDS Page, Incubation, Mass Spectrometry, Generated, Protein Binding, Labeling

Journal: bioRxiv

Article Title: A conserved mechanism for the retrieval of polyubiquitinated proteins from cilia

doi: 10.1101/2025.04.24.650332

Figure Lengend Snippet: Related to . (A)A simplified phylogenetic tree illustrating the evolutionary relationships among various species, with indicators for the presence of cilia/flagella, ubiquitin, intraflagellar transport (IFT) proteins, and CFAP36. CFAP36 is only present in organisms with cilia, ubiquitin and IFT. (B)A multiple sequence alignment of CFAP36 protein sequences from the five organisms used in this study: Homo sapiens (human), Sus scrofa (porcine), Mus musculus (murine), Chlamydomonas reinhardtii (green algae), and Leishmania tarentolae (protozoan parasite). Secondary structure elements predicted from the human CFAP36 sequence are annotated above the alignment, with α-helices (α) indicated. The BART-like domain and predicted ubiquitin interacting motifs (UIMs) are labeled. Invariant residues, conserved across all five species, are highlighted in rust. Similar residues, sharing conserved properties, are highlighted in yellow.

Article Snippet: Briefly, L. tarentolae strain P10 (Jena Bioscience, #LT-101) were grown in BHI medium (HIMEDIA, #N210) supplemented with 5 mg/mL hemin chloride (Sigma, #3741) and 10 U/mL Penicillin-Streptomycin (Gibco, #15070063) at 26 °C.

Techniques: Sequencing, Algae, Labeling

Journal: bioRxiv

Article Title: An inhibitor targeting glycosome membrane biogenesis kills Leishmania parasites

doi: 10.1101/2024.09.13.612636

Figure Lengend Snippet: Wild-type Leishmania tarentolae promastigotes (LtP) were treated with 2-fold serial dilution of inhibitors, starting at 100 μM. After 3 days of treatment, cell viability was assessed using a resazurin assay. The untreated control group was set as 100 % viability, and treated groups were normalized to this control. The EC 50 (half maximal effective concentration) values of compounds were determined as follows: 3.9 μM (Compound 1), 22.1 μM (Compound 2), 16.4 μM (Compound 3), 14.6 μM (Compound 4), 53.1 μM (Compound 5), and 23.4 μM (Compound 6). The results were plotted and analyzed using a dose-response model (EC50 shift, X is concentration) in GraphPad Prism 10. Every data point represents the mean of three biological replicates, and the error bars indicate the standard deviations. Among the six compounds, Compound 1 exhibited the strongest anti-leishmanial effect, while Compound 5 was the least effective.

Article Snippet: L. tarentolae promastigotes (LEXSY host P10, Jena Bioscience) were used in this study.

Techniques: Serial Dilution, Resazurin Assay, Control, Concentration Assay

Journal: bioRxiv

Article Title: An inhibitor targeting glycosome membrane biogenesis kills Leishmania parasites

doi: 10.1101/2024.09.13.612636

Figure Lengend Snippet: A) Wild-type L. tarentolae promastigotes (LtP) were treated with either DMSO(-) or 12 μM of Compound 1 (+) for 24 h. Digitonin fractionation was performed to investigate the mislocalization of glycosome matrix proteins. Enolase was used as cytosolic marker. The PTS1 proteins glycerol kinase (GK) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) served as glycosome matrix markers for PTS1 import, while aldolase served as a marker for glycosomal PTS2 import. The mitochondrial heat shock protein (mtHSP70) was used as a mitochondrial matrix marker to confirm that mitochondria were unaffected. Compared to the untreated group, GK, GAPDH, and aldolase were released from Compound 1-treated cells at lower levels of digitonin. The result indicated a partial mislocalization of glycosomal matrix proteins to cytosol upon Compound 1 treatment. B) LtP cells treated with DMSO or Compound 1 were fixed after 24 h of incubation and stained with anti- Tb Aldolase primary antibody. The red punctuate staining indicates the glycosomal localization of aldolase. Compared to the DMSO control group, cells treated with Compound 1 showed bigger red puncta, which might be related to enlarged or cluster glycosomes, suggesting a mild effect of Compound 1 on glycosomes. C) Human T-REx-293 cells were incubated with three concentrations of Compound 1 for 48 or 72 h and analyzed via immunoblotting using 3-ketoacyl-CoA thiolase (Thiolase) primary antibody. Precursor thiolase (p-Thiolase) is proteolytically processed upon peroxisomal import into its mature form (m-Thiolase), which runs at 44 kDa and serves as an indicator for peroxisomal import. Compound 1-treated wild-type (WT) cells displayed an m-Thiolase pattern similar to DMSO-treated cells, indicating that the peroxisomal protein import is normal. PEX3- and PEX19-knockout (KO) cells, in which p-Thiolase is abundant due to the absence of peroxisomes, were used as controls. D) Human T-REx293 cells, PEX3-KO, and PEX19-KO cells were grown on coverslips and treated with DMSO or 12 μM of Compound 1 for 72 h. Peroxisomes were labeled using anti-PMP70 antibody, whereas nuclei were stained with DAPI. Compound 1-treated cells showed a peroxisome pattern like that of the control (DMSO-treated) cells, indicating normal peroxisome morphology. No peroxisomes were observed in PEX3-KO and PEX19-KO cells.

Article Snippet: L. tarentolae promastigotes (LEXSY host P10, Jena Bioscience) were used in this study.

Techniques: Fractionation, Marker, Incubation, Staining, Control, Western Blot, Knock-Out, Labeling

Journal: Microbial Cell

Article Title: A Modular Cloning Toolkit for the production of recombinant proteins in Leishmania tarentolae

doi: 10.15698/mic2024.04.821

Figure Lengend Snippet: (A) Map of the destination vector for the L. tarentolae MoClo kit based on the pLEXSY_I-blecherry3 plasmid from Jena Bioscience. BsaI restriction sites were removed and the coding region for lacZα flanked by BsaI restriction sites was introduced to yield CCAT and GCTT fusion sites upon digestion with BsaI. (B) List of 34 Level 0 MoClo parts for positions B2 to B5 that are compatible with the MoClo syntax for plants and algae. The color code for fusion sites was adopted from the Chlamydomonas MoClo kit . Nucleotides used in codons are underlined in white. SP, signal peptide; CDS, coding sequence; RS, retention signal; sAP1, secreted acid phosphatase 1; HA, human influenza hemagglutinin; Met, methionine; Strep, streptavidin; PP, PreScission protease cleavage site; GST, glutathione transferase; RBD, receptor binding domain of SARS-CoV-2; SV40, simian-virus 40; TEV, tobacco etch virus protease cleavage site; SP, serine-proline repeat; ER, endoplasmic reticulum.

Article Snippet: L. tarentolae strain T7-TR promastigotes (Jena Bioscience) were cultured at 27°C in ventilated tissue culture flasks in an upright position on a Rotamax 120 shaker at 50 rpm in 10 mL brain heart infusion (BHI) medium according to standard protocols [ , , ].

Techniques: Plasmid Preparation, Algae, Sequencing, Binding Assay, Virus

Journal: Microbial Cell

Article Title: A Modular Cloning Toolkit for the production of recombinant proteins in Leishmania tarentolae

doi: 10.15698/mic2024.04.821

Figure Lengend Snippet: List of level 0 parts for the L. tarentolae MoClo system.

Article Snippet: L. tarentolae strain T7-TR promastigotes (Jena Bioscience) were cultured at 27°C in ventilated tissue culture flasks in an upright position on a Rotamax 120 shaker at 50 rpm in 10 mL brain heart infusion (BHI) medium according to standard protocols [ , , ].

Techniques: Sequencing, Protein Purification, Strep-tag, Binding Assay, Amplification, Luciferase, Purification

Journal: Microbial Cell

Article Title: A Modular Cloning Toolkit for the production of recombinant proteins in Leishmania tarentolae

doi: 10.15698/mic2024.04.821

Figure Lengend Snippet: (A) Schematic overview and expected mass of the secreted RBD fusion proteins. (B) Comparative Western blot analysis with antibodies against the RBD domain (left) and the GST domain (right) of RBD fusion proteins in the cell-containing pellet fraction (P) and supernatant fraction (S) following tetracycline induction of the according L. tarentolae liquid cultures. Recombinant RBD served as positive control (p.c.) and an induced culture without plasmid as negative control (n.c.). The calculated masses from panel A are indicated.

Article Snippet: L. tarentolae strain T7-TR promastigotes (Jena Bioscience) were cultured at 27°C in ventilated tissue culture flasks in an upright position on a Rotamax 120 shaker at 50 rpm in 10 mL brain heart infusion (BHI) medium according to standard protocols [ , , ].

Techniques: Western Blot, Recombinant, Positive Control, Plasmid Preparation, Negative Control

Journal: Microbial Cell

Article Title: A Modular Cloning Toolkit for the production of recombinant proteins in Leishmania tarentolae

doi: 10.15698/mic2024.04.821

Figure Lengend Snippet: (A) Schematic overview and expected mass of the secreted RBD variants with swapped C-terminal peptide tags. Variant 2 with an N-terminal 3xHA tag instead of the N-terminal signal peptide served as a cytosolic control. Variant 4 also contains an SP20 sequence for extensive O -glycosylation. (B) Comparative Western blot analysis with antibodies against the RBD domain (left) or the 8xHis tag (right) in the cell-containing pellet fraction (P) and supernatant fraction (S) following tetracycline induction of the according L. tarentolae liquid cultures. An induced culture without plasmid served as negative control (n.c.) and recombinant His-tagged RBD with an expected size of 35 kDa as positive control (p.c.). The asterisk labels a band that was caused by a contamination of the supernatant fraction in the experiment shown. Both membranes were stripped and redecorated with an antibody against the HA tag. The calculated masses from panel A are indicated.

Article Snippet: L. tarentolae strain T7-TR promastigotes (Jena Bioscience) were cultured at 27°C in ventilated tissue culture flasks in an upright position on a Rotamax 120 shaker at 50 rpm in 10 mL brain heart infusion (BHI) medium according to standard protocols [ , , ].

Techniques: Variant Assay, Sequencing, Western Blot, Plasmid Preparation, Negative Control, Recombinant, Positive Control

Journal: Microbial Cell

Article Title: A Modular Cloning Toolkit for the production of recombinant proteins in Leishmania tarentolae

doi: 10.15698/mic2024.04.821

Figure Lengend Snippet: (A) Schematic overview and expected mass of the secreted RBD fusion proteins. (B) Western blot analysis with an antibody against the RBD domain of RBD fusion variants 1 and 2 in the cell-containing pellet fraction (P) and supernatant fraction (S) following tetracycline induction of the according L. tarentolae liquid cultures for the indicated time periods. An induced culture without plasmid served as negative control (n.c.) and recombinant RBD as positive control (p.c.). The calculated masses from panel A are indicated. (C) Western blot analysis with antibodies against the C-terminal His tag (left) or the RBD domain (right) of fusion variant 3.

Article Snippet: L. tarentolae strain T7-TR promastigotes (Jena Bioscience) were cultured at 27°C in ventilated tissue culture flasks in an upright position on a Rotamax 120 shaker at 50 rpm in 10 mL brain heart infusion (BHI) medium according to standard protocols [ , , ].

Techniques: Western Blot, Plasmid Preparation, Negative Control, Recombinant, Positive Control, Variant Assay

Journal: Microbial Cell

Article Title: A Modular Cloning Toolkit for the production of recombinant proteins in Leishmania tarentolae

doi: 10.15698/mic2024.04.821

Figure Lengend Snippet: (A) Schematic overview and expected mass of the secreted RBD fusion proteins. (B) Western blot analysis with antibodies against the RBD domain (left) and the GST domain (right) of RBD fusion variant 1 in the cell-containing pellet fraction (P) and supernatant fraction (S) following tetracycline induction of the according L. tarentolae liquid cultures containing the indicated amounts of antibiotics. An induced culture without plasmid served as negative control (n.c.) and recombinant RBD as positive control (p.c.). The asterisk labels the TCA precipitate of the 50% supernatant fraction which was lost in the experiment shown. The calculated mass from panel A is indicated. (C) Western blot analysis with antibodies against the HA tag (left), the RBD domain (right), and the His tag (bottom) of RBD fusion variant 2.

Article Snippet: L. tarentolae strain T7-TR promastigotes (Jena Bioscience) were cultured at 27°C in ventilated tissue culture flasks in an upright position on a Rotamax 120 shaker at 50 rpm in 10 mL brain heart infusion (BHI) medium according to standard protocols [ , , ].

Techniques: Western Blot, Variant Assay, Plasmid Preparation, Negative Control, Recombinant, Positive Control

Journal: The Journal of Biological Chemistry

Article Title: Resolving the polycistronic aftermath: Essential role of topoisomerase IA in preventing R-loops in L eishmania

doi: 10.1016/j.jbc.2024.107162

Figure Lengend Snippet: Topoisomerase IA, an essential protein of L. donovani . A , sequence comparison of E. coli TOPIA, M. tuberculosis TOPIA, T. brucei TOPIA and L. donovani TOPIA showing start and end residue of TOPRIM domain, Active site tyrosine and DNA binding domain region. B , homology modeled structure of LdTOPIA with the active site residues Tyr357, Glu135, Asp131, and Asp 133 exhibited in the zoomed image. C , microscopic images of (−Tet) tetracycline uninduced (top) and (+Tet) induced ( bottom ) LtT7TR parasites expressing antisense LdTOPIA construct, Scale bar: 25 μm (ii) Graphical representation of percentage viable LtT7TR parasites in (−Tet) and (+Tet) condition for indicated time points. (n = 5 mean ± SD, 3 biological replicates. p vs. respective control (0h)). D , relative quantitation of LtTOPIA, LtTOPIL, and Ltβ-Tub mRNA expression levels in (+Tet) parasites measured by qPCR and plotted as normalized values over 24 h (n = 3 mean ± SD, 3 biological replicates. p versus tetracycline treated for 24 h). E , flow cytometric analysis of cell-cycle arrest in antisense LtTOPIA transfected LtT7TR parasites without (−Tet, green ) or with (+Tet, red ) induction at indicated timepoints (representative image of n = 3). F , graphical representation of the cell cycle phases (G 0 -G 1 , S, G 2- M, and 4N) for antisense LtTOPIA transfected LtT7TR parasites without (−Tet) or with (+Tet) induction for indicated time points (n = 5, mean ± SD, 3 biological replicates for each time).

Article Snippet: The conditional antisense strategy uses a commercially available L. tarentolae (LtT7TR) strain (Jena Bioscience) wherein the tetracycline repressor (TR) and the T7 RNA polymerase (T7) is integrated into the genome of this strain under the control of hygromycin and nourseothricin selectable markers, respectively ( E -i) ( , ).

Techniques: Sequencing, Comparison, Residue, Binding Assay, Expressing, Construct, Quantitation Assay, Transfection

Journal: The Journal of Biological Chemistry

Article Title: Resolving the polycistronic aftermath: Essential role of topoisomerase IA in preventing R-loops in L eishmania

doi: 10.1016/j.jbc.2024.107162

Figure Lengend Snippet: Functional characterization of purified LdTOPIA. A , SDS-PAGE (10%) analysis of purified LdTOPIA, LdTOPIA Y357A , and LdTOPIA E135A from tetracycline induced, pLew100v5 cloned LdTOPIA, LdTOPIA Y357A , and LdTOPIA E135A transfected LtT7TR conditional expression system, stained with Coomassie G-250. Plasmid DNA relaxation assay using (−SC) pBluescript and ( B ) LdTOPIA or its active site mutants LdTOPIA Y357A and LdTOPIA E135A or ( C ) LdTOPIA along with Camptothecin (CPT) or Etoposide (Etop) and in presence of Mg 2+ at 37 °C for 25 min followed by electrophoresis in 1% agarose gel and thereafter EtBr staining for visualization. D , plasmid DNA relaxation assay using (−SC) pBluescript, increasing concentration of Mg 2+ and purified LdTOPIA at 37 °C for 15 min. E , bidirectional agarose gel electrophoresis using (−SC) pBluescript and reverse gyrase generated (+SC) pBluescript plasmid DNA incubated with Human TOPII and purified LdTOPIA in order to differentiate the relaxation of negative and positive topoisomers. F , electrophoretic mobility shift assay (EMSA) using 100 nM γ-32P labeled (i) single-stranded and (ii) double-stranded oligonucleotide substrates incubated with increasing concentrations of LdTOPIA (5–200) nM ( G ) DNA binding affinity was measured by fluorescence polarization using 5′ FAM tagged ssDNA and dsDNA substrate incubated with increasing concentration of LdTOPIA. Fraction-bound values were plotted against LdTOPIA concentration (2–200) nM and K D values of LdTOPIA were calculated for ssDNA and dsDNA (n = 5, mean ± SD, 3 biological replicates).

Article Snippet: The conditional antisense strategy uses a commercially available L. tarentolae (LtT7TR) strain (Jena Bioscience) wherein the tetracycline repressor (TR) and the T7 RNA polymerase (T7) is integrated into the genome of this strain under the control of hygromycin and nourseothricin selectable markers, respectively ( E -i) ( , ).

Techniques: Functional Assay, Purification, SDS Page, Clone Assay, Transfection, Expressing, Staining, Plasmid Preparation, Electrophoresis, Agarose Gel Electrophoresis, Concentration Assay, Generated, Incubation, Electrophoretic Mobility Shift Assay, Labeling, Binding Assay, Fluorescence

Journal: The Journal of Biological Chemistry

Article Title: Resolving the polycistronic aftermath: Essential role of topoisomerase IA in preventing R-loops in L eishmania

doi: 10.1016/j.jbc.2024.107162

Figure Lengend Snippet: LdTOPIA prevents R-loop formation during polycistronic transcription. A , LtT7TR strain transfected with anti-LtTOPIA construct was either untreated or treated with tetracycline for indicated time points to observe the extent of R-loop formation. Parasites were stained with anti-LdTOPIA-AlexaFluor488, anti-DNA-RNA (S9.6) Alexa Fluor568 antibodies, and counterstained with DRAQ5. Scale Bar, 5 μm. B , graphical representation of the extent of R-loop formation estimated from the fluorescence intensity. [n = 60 (20 nuclei of 3 biological replicates), mean ± SD. p versus 0h]. C , DRIB assay was carried out from genomic DNA isolated from untreated or tetracycline-treated antisense LtTOPIA transfected LtT7TR parasites after indicated time points. D , densitometry of the same samples. [n = 3 and 3 biological replicates, mean ± SD, p values for (+Tet) versus (+Tet +RNaseH) and p versus 0h of (+Tet +RNaseH)]. E , DRIB assay was carried out using genomic DNA isolated from untreated or tetracycline-treated antisense LtTOPIA transfected LtT7TR parasites and complemented with LdTOPIA, LdTOPIAΔNLS or LdTOPIAΔNLS-SV40NLS for indicated time points. F , densitometric analysis of the same samples [n = 3 and 3 biological replicates, mean ± SD, p values for (+Tet) versus (+Tet +LdTOPIA) and p versus 0h of (+Tet +LdTOPIA)].

Article Snippet: The conditional antisense strategy uses a commercially available L. tarentolae (LtT7TR) strain (Jena Bioscience) wherein the tetracycline repressor (TR) and the T7 RNA polymerase (T7) is integrated into the genome of this strain under the control of hygromycin and nourseothricin selectable markers, respectively ( E -i) ( , ).

Techniques: Transfection, Construct, Staining, Fluorescence, Isolation

Journal: Biomedicines

Article Title: Systematic Exploration of Functional Group Relevance for Anti-Leishmanial Activity of Anisomycin

doi: 10.3390/biomedicines11092541

Figure Lengend Snippet: Inhibition of translation in active cell lysates derived from L. tarentolae , bacteria ( E. coli ), and rabbit reticulocytes (RR) and inhibition of L. donovani promastigote growth by anisomycin and derivatives.

Article Snippet: Cytosolic ribosome susceptibility and selectivity were assessed through three different cell-free transcription-translation assays: (1) An extract of E. coli (S30, Promega, Madison, WI, USA), (2) in rabbit reticulocytes (Promega), and (3) in L. tarentolae lysate (Jena Bioscience, Jena, Germany).

Techniques: Inhibition, Derivative Assay, Bacteria