2014
DOI: 10.1590/0074-0276140238
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Abstract: We present here three expression plasmids for Trypanosoma cruzi adapted to the Gateway® recombination cloning system. Two of these plasmids were designed to express trypanosomal proteins fused to a double tag for tandem affinity purification (TAPtag). The TAPtag and Gateway® cassette were introduced into an episomal (pTEX) and an integrative (pTREX) plasmid. Both plasmids were assayed by introducing green fluorescent protein (GFP) by recombination and the integrity of the double-tagged protein was determined b… Show more

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Cited by 18 publications
(24 citation statements)
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(17 reference statements)
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“…Overexpression of Tc SIR2RP1 and Tc SIR2RP3 enzymes was performed using the T . cruzi inducible vector p Tc INDEXGW [ 33 ]. Epimastigote cell lines expressing each sirtuin with a C-terminal HA tag under the control of a Tetracycline-regulated promoter were generated (Materials and Methods).…”
Section: Resultsmentioning
confidence: 99%
“…Overexpression of Tc SIR2RP1 and Tc SIR2RP3 enzymes was performed using the T . cruzi inducible vector p Tc INDEXGW [ 33 ]. Epimastigote cell lines expressing each sirtuin with a C-terminal HA tag under the control of a Tetracycline-regulated promoter were generated (Materials and Methods).…”
Section: Resultsmentioning
confidence: 99%
“…Then, the inserts were transferred by recombination using LR clonase II enzyme mix (Invitrogen) to T . cruzi expression vector p Tc INDEX-GW [ 27 ]—an integrative vector derivate of p Tc INDEX [ 28 ] containing an inducible expression site under tetracycline control. For yeast assays, the same inserts were cloned in the p426.M25 inducible vector under the control of the MET25 promoter [ 29 ], previously used in our laboratory [ 30 ].…”
Section: Methodsmentioning
confidence: 99%
“…A Gateway® cloning‐compatible (Alonso, Ritagliati, Cribb, & Serra, ) pTcINDEX system was used to express Tc RND integrated into the transcriptionally silent rRNA spacer region in T. cruzi constitutively expressing T7 polymerase and tet repressor episomally (Taylor & Kelly, ). Experiments were performed in two backgrounds.…”
Section: Resultsmentioning
confidence: 99%
“…The Tc RND nucleotide sequence was amplified by PCR from CL Brener strain genomic DNA using the oligonucleotides Tc RND‐Fwd (5ʹ‐TTCAGTCGACTGGATCCATGCTGCGTCGTGYTGGTGTC‐3ʹ) and Tc RND‐Rev (5ʹ‐AAAGTCGGGTCTAGATATCTCAAGAAGAGGATCTTTCGTCAAAAAAG‐3ʹ) designed to amplify either allele (TcCLB.510743.90 or TcCLB.510659.219). The PCR product was cloned into pJET1.2 (Thermo Scientific) and excised at BamHI and EcoRV sites to be inserted in these sites in pENTR4 (Invitrogen), and then transferred to p Tc INDEX‐GW (Alonso et al, ) by recombination using LR clonase II enzyme mix (Invitrogen) per manufacturer instructions to generate p Tc INDEX‐GW‐ Tc RND.…”
Section: Methodsmentioning
confidence: 99%