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A high-throughput cloning system for reverse genetics in Trypanosoma cruzi.

Batista M, Marchini FK, Celedon PA, Fragoso SP, Probst CM, Preti H, Ozaki LS, Buck GA, Goldenberg S, Krieger MA - BMC Microbiol. (2010)

Bottom Line: The vectors were verified by successful subcellular localization of two previously characterized proteins (TcRab7 and PAR 2) and a putative centrin.This platform also allows vector customization, as the vectors were constructed to enable easy exchange of its elements.The development of this high-throughput platform is a step closer towards large-scale trypanosome applications and initiatives.

View Article: PubMed Central - HTML - PubMed

Affiliation: Instituto Carlos Chagas, FIOCRUZ, Curitiba, Parana, Brazil.

ABSTRACT

Background: The three trypanosomatids pathogenic to men, Trypanosoma cruzi, Trypanosoma brucei and Leishmania major, are etiological agents of Chagas disease, African sleeping sickness and cutaneous leishmaniasis, respectively. The complete sequencing of these trypanosomatid genomes represented a breakthrough in the understanding of these organisms. Genome sequencing is a step towards solving the parasite biology puzzle, as there are a high percentage of genes encoding proteins without functional annotation. Also, technical limitations in protein expression in heterologous systems reinforce the evident need for the development of a high-throughput reverse genetics platform. Ideally, such platform would lead to efficient cloning and compatibility with various approaches. Thus, we aimed to construct a highly efficient cloning platform compatible with plasmid vectors that are suitable for various approaches.

Results: We constructed a platform with a flexible structure allowing the exchange of various elements, such as promoters, fusion tags, intergenic regions or resistance markers. This platform is based on Gateway® technology, to ensure a fast and efficient cloning system. We obtained plasmid vectors carrying genes for fluorescent proteins (green, cyan or yellow), and sequences for the c-myc epitope, and tandem affinity purification or polyhistidine tags. The vectors were verified by successful subcellular localization of two previously characterized proteins (TcRab7 and PAR 2) and a putative centrin. For the tandem affinity purification tag, the purification of two protein complexes (ribosome and proteasome) was performed.

Conclusions: We constructed plasmids with an efficient cloning system and suitable for use across various applications, such as protein localization and co-localization, protein partner identification and protein expression. This platform also allows vector customization, as the vectors were constructed to enable easy exchange of its elements. The development of this high-throughput platform is a step closer towards large-scale trypanosome applications and initiatives.

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Related in: MedlinePlus

Detection of GFP-fused recombinant proteins and FACS analysis. Lanes in A and B represent protein extracts from T. cruzi wild type (WT) cells and cells transfected with GFPneo-CTRL, GFPneo-Rab7 and GFPneo-PAR2. In A is represented the load control gel. In B, these extracts were incubated with antibodies against GFP. BenchMark (Invitrogen) was used as the molecular weight marker. In C, T. cruzi wild type epimastigotes (WT) were used as a negative control. For each culture, 20,000 cells were counted. The Y- and X-axis represent the number of cells counted (events) and GFP fluorescence (FL1-H) in arbitrary fluorescence units (AFU), respectively.
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Figure 3: Detection of GFP-fused recombinant proteins and FACS analysis. Lanes in A and B represent protein extracts from T. cruzi wild type (WT) cells and cells transfected with GFPneo-CTRL, GFPneo-Rab7 and GFPneo-PAR2. In A is represented the load control gel. In B, these extracts were incubated with antibodies against GFP. BenchMark (Invitrogen) was used as the molecular weight marker. In C, T. cruzi wild type epimastigotes (WT) were used as a negative control. For each culture, 20,000 cells were counted. The Y- and X-axis represent the number of cells counted (events) and GFP fluorescence (FL1-H) in arbitrary fluorescence units (AFU), respectively.

Mentions: To confirm the presence of recombinant proteins in transfected T. cruzi, western blot assays were performed using antibodies against the tags. The bands in Figure 3B correspond to the expected molecular weight of the PAR 2 and TcRab7 with addition of the GFP tag and the sequence for the attB1 site. Detection of TcrL27 and Tcpr29A recombinant proteins (using anti-calmodulin binding peptide antibody) is shown in the "Tandem affinity purification" section, while the centrin recombinant protein used with c-myc and polyhistidine tags (using anti-c-myc and anti-histidine antibodies) are shown in Additional file 1 - Figure S1. Predicted molecular weight of native proteins TcrL27, Tcpr29A, PAR 2, centrin and TcRab7, including the protein tags are described in Additional file 2 - Table S1.


A high-throughput cloning system for reverse genetics in Trypanosoma cruzi.

Batista M, Marchini FK, Celedon PA, Fragoso SP, Probst CM, Preti H, Ozaki LS, Buck GA, Goldenberg S, Krieger MA - BMC Microbiol. (2010)

Detection of GFP-fused recombinant proteins and FACS analysis. Lanes in A and B represent protein extracts from T. cruzi wild type (WT) cells and cells transfected with GFPneo-CTRL, GFPneo-Rab7 and GFPneo-PAR2. In A is represented the load control gel. In B, these extracts were incubated with antibodies against GFP. BenchMark (Invitrogen) was used as the molecular weight marker. In C, T. cruzi wild type epimastigotes (WT) were used as a negative control. For each culture, 20,000 cells were counted. The Y- and X-axis represent the number of cells counted (events) and GFP fluorescence (FL1-H) in arbitrary fluorescence units (AFU), respectively.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3020659&req=5

Figure 3: Detection of GFP-fused recombinant proteins and FACS analysis. Lanes in A and B represent protein extracts from T. cruzi wild type (WT) cells and cells transfected with GFPneo-CTRL, GFPneo-Rab7 and GFPneo-PAR2. In A is represented the load control gel. In B, these extracts were incubated with antibodies against GFP. BenchMark (Invitrogen) was used as the molecular weight marker. In C, T. cruzi wild type epimastigotes (WT) were used as a negative control. For each culture, 20,000 cells were counted. The Y- and X-axis represent the number of cells counted (events) and GFP fluorescence (FL1-H) in arbitrary fluorescence units (AFU), respectively.
Mentions: To confirm the presence of recombinant proteins in transfected T. cruzi, western blot assays were performed using antibodies against the tags. The bands in Figure 3B correspond to the expected molecular weight of the PAR 2 and TcRab7 with addition of the GFP tag and the sequence for the attB1 site. Detection of TcrL27 and Tcpr29A recombinant proteins (using anti-calmodulin binding peptide antibody) is shown in the "Tandem affinity purification" section, while the centrin recombinant protein used with c-myc and polyhistidine tags (using anti-c-myc and anti-histidine antibodies) are shown in Additional file 1 - Figure S1. Predicted molecular weight of native proteins TcrL27, Tcpr29A, PAR 2, centrin and TcRab7, including the protein tags are described in Additional file 2 - Table S1.

Bottom Line: The vectors were verified by successful subcellular localization of two previously characterized proteins (TcRab7 and PAR 2) and a putative centrin.This platform also allows vector customization, as the vectors were constructed to enable easy exchange of its elements.The development of this high-throughput platform is a step closer towards large-scale trypanosome applications and initiatives.

View Article: PubMed Central - HTML - PubMed

Affiliation: Instituto Carlos Chagas, FIOCRUZ, Curitiba, Parana, Brazil.

ABSTRACT

Background: The three trypanosomatids pathogenic to men, Trypanosoma cruzi, Trypanosoma brucei and Leishmania major, are etiological agents of Chagas disease, African sleeping sickness and cutaneous leishmaniasis, respectively. The complete sequencing of these trypanosomatid genomes represented a breakthrough in the understanding of these organisms. Genome sequencing is a step towards solving the parasite biology puzzle, as there are a high percentage of genes encoding proteins without functional annotation. Also, technical limitations in protein expression in heterologous systems reinforce the evident need for the development of a high-throughput reverse genetics platform. Ideally, such platform would lead to efficient cloning and compatibility with various approaches. Thus, we aimed to construct a highly efficient cloning platform compatible with plasmid vectors that are suitable for various approaches.

Results: We constructed a platform with a flexible structure allowing the exchange of various elements, such as promoters, fusion tags, intergenic regions or resistance markers. This platform is based on Gateway® technology, to ensure a fast and efficient cloning system. We obtained plasmid vectors carrying genes for fluorescent proteins (green, cyan or yellow), and sequences for the c-myc epitope, and tandem affinity purification or polyhistidine tags. The vectors were verified by successful subcellular localization of two previously characterized proteins (TcRab7 and PAR 2) and a putative centrin. For the tandem affinity purification tag, the purification of two protein complexes (ribosome and proteasome) was performed.

Conclusions: We constructed plasmids with an efficient cloning system and suitable for use across various applications, such as protein localization and co-localization, protein partner identification and protein expression. This platform also allows vector customization, as the vectors were constructed to enable easy exchange of its elements. The development of this high-throughput platform is a step closer towards large-scale trypanosome applications and initiatives.

Show MeSH
Related in: MedlinePlus