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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

Efficiency of L27 and 29A complexes purification with the original TAP tag tested in T. cruzi cells. In A, the TAP tag-fused TcrL27 (L27), Tcpr29A (29A) and the control TAPneo-CTRL (CTRL) was detected by western blot with anti-CBP antibody. In B, the fractions from TAP purification were probed with anti-L26 and anti-α2 in immunoblots. Lanes represent total protein (T) or eluted product after digestion (E). BenchMark (Invitrogen) was used as the molecular weight marker.
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Figure 5: Efficiency of L27 and 29A complexes purification with the original TAP tag tested in T. cruzi cells. In A, the TAP tag-fused TcrL27 (L27), Tcpr29A (29A) and the control TAPneo-CTRL (CTRL) was detected by western blot with anti-CBP antibody. In B, the fractions from TAP purification were probed with anti-L26 and anti-α2 in immunoblots. Lanes represent total protein (T) or eluted product after digestion (E). BenchMark (Invitrogen) was used as the molecular weight marker.

Mentions: The tandem affinity purification (TAP) tag [40] comprises two repeated B domain of protein A (able to bind IgG), plus the site for TEV protease and the calmodulin binding peptide (CBP). The main reason for using a tandem purification approach is to avoid false positives. Two genes already described in the literature, Tcpr29A [41] and TcrL27 [42] were inserted into pTcTAPN. TcrL27 encodes the L27 protein, a member of the larger ribosomal subunit, and Tcpr29A (29A) is a gene encoding the α6 20S proteasome subunit. The TAP tag-fused L27, 29A and the control TAPneo-CTRL (CTRL) were detected by western blot with anti-CBP antibody (Figure 5A).


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)

Efficiency of L27 and 29A complexes purification with the original TAP tag tested in T. cruzi cells. In A, the TAP tag-fused TcrL27 (L27), Tcpr29A (29A) and the control TAPneo-CTRL (CTRL) was detected by western blot with anti-CBP antibody. In B, the fractions from TAP purification were probed with anti-L26 and anti-α2 in immunoblots. Lanes represent total protein (T) or eluted product after digestion (E). BenchMark (Invitrogen) was used as the molecular weight marker.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 5: Efficiency of L27 and 29A complexes purification with the original TAP tag tested in T. cruzi cells. In A, the TAP tag-fused TcrL27 (L27), Tcpr29A (29A) and the control TAPneo-CTRL (CTRL) was detected by western blot with anti-CBP antibody. In B, the fractions from TAP purification were probed with anti-L26 and anti-α2 in immunoblots. Lanes represent total protein (T) or eluted product after digestion (E). BenchMark (Invitrogen) was used as the molecular weight marker.
Mentions: The tandem affinity purification (TAP) tag [40] comprises two repeated B domain of protein A (able to bind IgG), plus the site for TEV protease and the calmodulin binding peptide (CBP). The main reason for using a tandem purification approach is to avoid false positives. Two genes already described in the literature, Tcpr29A [41] and TcrL27 [42] were inserted into pTcTAPN. TcrL27 encodes the L27 protein, a member of the larger ribosomal subunit, and Tcpr29A (29A) is a gene encoding the α6 20S proteasome subunit. The TAP tag-fused L27, 29A and the control TAPneo-CTRL (CTRL) were detected by western blot with anti-CBP antibody (Figure 5A).

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