Construction of three new Gateway® expression
 plasmids for Trypanosoma cruzi

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 by western blotting and immunofluorescence microscopy. The third Gateway adapted vector assayed was the inducible pTcINDEX. When tested with GFP, pTcINDEX-GW showed a good response to tetracycline, being less leaky than its precursor (pTcINDEX).

Trypanosoma cruzi; expression vectors - Gateway^® system; tandem affinity purification; pTcINDEX

Authorship

Victoria L Alonso

Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario, Rosario, ArgentinaUniversidad Nacional de RosarioArgentinaRosario, ArgentinaFacultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario, Rosario, Argentina

Instituto de Biología Molecular y Celular de Rosario, Consejo Nacional de Investigaciones Científicas y Técnicas, Rosario, ArgentinaConsejo Nacional de Investigaciones Científicas y TécnicasArgentinaRosario, ArgentinaInstituto de Biología Molecular y Celular de Rosario, Consejo Nacional de Investigaciones Científicas y Técnicas, Rosario, Argentina

Carla Ritagliati

Pamela Cribb

Esteban C Serra ⁺+ Corresponding author: eserra@fbioyf.unr.edu.ar

+ Corresponding author: eserra@fbioyf.unr.edu.ar

SCIMAGO INSTITUTIONS RANKINGS

Figures

Figures (3)

Thumbnail

Fig. 1
: restriction maps and genetic elements of the Gateway® plasmids (A) pTEX-TAPtag-GW map. The ampicillin resistance gene (neo r), Gateway® cassette and TAPtag sequence are flanked by the 5’-upstream and 3-downstream regions of the Trypanosoma cruzi glycosomal glycosomal glyceraldehyde-3-phosphate dehydrogenase gene (gGAPDH) genes. The Gateway® cassette consists of the attR1 and R2 sites (black boxes), the chloramphenicol resistance gene (Cmr) and the ccdB gene. The TAPtag cassette consists of the calmodulin-binding peptide (CBP), protein A (PA) and a spacer region with a Tobacco Etch Virus (TEV) protease cleavage site; B: pTREX-TAPtag-GW map. This vector has the same features as pTEX with the addition of a ribosomal promoter (with a transcription starting point, black arrow head) and the HX1 fragment; C: inducible expression vector pTcINDEX-GW. The grey box indicates the HX1 fragment. The T. cruzi actin intergenic region (TcActin IG), the T7 transcriptional terminator (T) and the Gateway® cassette are shown. The black flags indicate the T7 promoter and the grey circle denotes the location of the tetracycline operator. R-NTS/P is the ribosomal non-transcribed spacer and promoter region used for targeting. The Roman numerals I and II indicate the two halves of the targeting sequence cloned in the opposite orientation of that in the genome. The black arrowhead indicates the location of the polymerase I transcription start site.

Thumbnail

Fig. 2
: pTEX and pTREX-TAPtag-GW experimental validation (A) Coomassie and western blot analysis of total lysates from CL Brener wild type cells (Lane 1) and those transfected (2) with pTEX-TAPtag-GW-GFP or pTREX-TAPtag-GW-GFP. Anti-green fluorescent protein (GFP) (a-GFP) and anti-protein A (a-protein A) antibodies were used. Bound antibodies were detected using peroxidase-labelled anti-mouse or anti-rabbit immunoglobulin G (IgG) (GE Healthcare) and ECL Prime (GE Healthcare) using standard protocols; B: immunofluorescence microscopy of CL Brener transfected with pTEX-TAPtag-GW-GFP and pTREX-TAPtag-GW-GFP. Parasites were adhered to poly-L-lysine coated slides and fixed with 4% paraformaldehyde in phosphate-buffered saline (PBS), permeabilised with 0.1% Triton X-100 in PBS and then incubated with a-protein A antibodies in 1% bovine serum albumin-PBS for 3 h. Then, the slides were incubated with anti-mouse IgG antibody conjugated to Alexa Fluor 555 secondary antibodies for 1 h. The slides were mounted with VectaShield (Vector Laboratories) in the presence of 2 µg mL-1 4’-6-diamidino-2-phenylindole (DAPI) in PBS. Images were acquired with a Nikon Eclipse TE-2000-E2 confocal microscope. Adobe Photoshop CS v.8.0.1 (Adobe System Incorporated) and Nikon EZ-C1 FreeViewer v.3.70 (Nikon Corporation) software were used to analyse the images.

Thumbnail

Fig. 3
: pTcINDEX-GW experimental validation. A: fluorescence microscopy of Dm28c epimastigotes transfected with pTcINDEX-GW-green fluorescent protein (GFP) and pTcINDEX-Red before and after induction with tetracycline (0.5 µg mL-1 for 96 h). Parasites were fixed with 4% paraformaldehyde in phosphate-buffered saline (PBS), permeabilised with 0.1% Triton X-100 in PBS and incubated with 2 µg mL-1 4’-6-diamidino-2-phenylindole (DAPI) in PBS before mounting the slides with VectaShield (Vector Laboratories). Images were acquired with a Nikon Eclipse TE-2000-E2 confocal microscope; B: western blot analysis of Dm28c pTcINDEX-GW-GFP total lysates induced with 0.5 µg mL-1 tetracycline for 0, 24, 48 and 72 h. Anti-GFP (a-GFP) and anti-α tubulin antibodies were used. Bound antibodies were detected with peroxidase-labelled anti-mouse or anti-rabbit immunoglobulin G (GE Healthcare) and ECL Prime (GE Healthcare) using standard protocols; C: growth curve of Dm28c pTcINDEX-GW-GFP induced with different concentrations of tetracycline (0-5 µg mL-1); D: expression of GFP after induction (0.5 µg mL-1 of tetracycline for 96 h) in Dm28c and CL Brener strains transfected with pTcINDEX-GW-GFP; E: fluorescence microscopy of Dm28c pTcINDEX-GW-GFP at different stages of the life cycle. Parasites were fixed with 4% paraformaldehyde in PBS, permeabilised with 0.1% Triton X-100 in PBS and incubated with 2 µg mL-1 DAPI in PBS before mounting the slides with VectaShield (Vector Laboratories). Images were acquired with a Nikon Eclipse E300 microscope.

Fig. 1 : restriction maps and genetic elements of the Gateway® plasmids (A) pTEX-TAPtag-GW map. The ampicillin resistance gene (neo r), Gateway® cassette and TAPtag sequence are flanked by the 5’-upstream and 3-downstream regions of the Trypanosoma cruzi glycosomal glycosomal glyceraldehyde-3-phosphate dehydrogenase gene (gGAPDH) genes. The Gateway® cassette consists of the attR1 and R2 sites (black boxes), the chloramphenicol resistance gene (Cmr) and the ccdB gene. The TAPtag cassette consists of the calmodulin-binding peptide (CBP), protein A (PA) and a spacer region with a Tobacco Etch Virus (TEV) protease cleavage site; B: pTREX-TAPtag-GW map. This vector has the same features as pTEX with the addition of a ribosomal promoter (with a transcription starting point, black arrow head) and the HX1 fragment; C: inducible expression vector pTcINDEX-GW. The grey box indicates the HX1 fragment. The T. cruzi actin intergenic region (TcActin IG), the T7 transcriptional terminator (T) and the Gateway® cassette are shown. The black flags indicate the T7 promoter and the grey circle denotes the location of the tetracycline operator. R-NTS/P is the ribosomal non-transcribed spacer and promoter region used for targeting. The Roman numerals I and II indicate the two halves of the targeting sequence cloned in the opposite orientation of that in the genome. The black arrowhead indicates the location of the polymerase I transcription start site.

Fig. 2 : pTEX and pTREX-TAPtag-GW experimental validation (A) Coomassie and western blot analysis of total lysates from CL Brener wild type cells (Lane 1) and those transfected (2) with pTEX-TAPtag-GW-GFP or pTREX-TAPtag-GW-GFP. Anti-green fluorescent protein (GFP) (a-GFP) and anti-protein A (a-protein A) antibodies were used. Bound antibodies were detected using peroxidase-labelled anti-mouse or anti-rabbit immunoglobulin G (IgG) (GE Healthcare) and ECL Prime (GE Healthcare) using standard protocols; B: immunofluorescence microscopy of CL Brener transfected with pTEX-TAPtag-GW-GFP and pTREX-TAPtag-GW-GFP. Parasites were adhered to poly-L-lysine coated slides and fixed with 4% paraformaldehyde in phosphate-buffered saline (PBS), permeabilised with 0.1% Triton X-100 in PBS and then incubated with a-protein A antibodies in 1% bovine serum albumin-PBS for 3 h. Then, the slides were incubated with anti-mouse IgG antibody conjugated to Alexa Fluor 555 secondary antibodies for 1 h. The slides were mounted with VectaShield (Vector Laboratories) in the presence of 2 µg mL-1 4’-6-diamidino-2-phenylindole (DAPI) in PBS. Images were acquired with a Nikon Eclipse TE-2000-E2 confocal microscope. Adobe Photoshop CS v.8.0.1 (Adobe System Incorporated) and Nikon EZ-C1 FreeViewer v.3.70 (Nikon Corporation) software were used to analyse the images.

Fig. 3 : pTcINDEX-GW experimental validation. A: fluorescence microscopy of Dm28c epimastigotes transfected with pTcINDEX-GW-green fluorescent protein (GFP) and pTcINDEX-Red before and after induction with tetracycline (0.5 µg mL-1 for 96 h). Parasites were fixed with 4% paraformaldehyde in phosphate-buffered saline (PBS), permeabilised with 0.1% Triton X-100 in PBS and incubated with 2 µg mL-1 4’-6-diamidino-2-phenylindole (DAPI) in PBS before mounting the slides with VectaShield (Vector Laboratories). Images were acquired with a Nikon Eclipse TE-2000-E2 confocal microscope; B: western blot analysis of Dm28c pTcINDEX-GW-GFP total lysates induced with 0.5 µg mL-1 tetracycline for 0, 24, 48 and 72 h. Anti-GFP (a-GFP) and anti-α tubulin antibodies were used. Bound antibodies were detected with peroxidase-labelled anti-mouse or anti-rabbit immunoglobulin G (GE Healthcare) and ECL Prime (GE Healthcare) using standard protocols; C: growth curve of Dm28c pTcINDEX-GW-GFP induced with different concentrations of tetracycline (0-5 µg mL-1); D: expression of GFP after induction (0.5 µg mL-1 of tetracycline for 96 h) in Dm28c and CL Brener strains transfected with pTcINDEX-GW-GFP; E: fluorescence microscopy of Dm28c pTcINDEX-GW-GFP at different stages of the life cycle. Parasites were fixed with 4% paraformaldehyde in PBS, permeabilised with 0.1% Triton X-100 in PBS and incubated with 2 µg mL-1 DAPI in PBS before mounting the slides with VectaShield (Vector Laboratories). Images were acquired with a Nikon Eclipse E300 microscope.

How to cite

copy

Instituto Oswaldo Cruz, Ministério da Saúde Av. Brasil, 4365 - Pavilhão Mourisco, Manguinhos, 21040-900 Rio de Janeiro RJ Brazil, Tel.: (55 21) 2562-1222, Fax: (55 21) 2562 1220 - Rio de Janeiro - RJ - Brazil
E-mail: memorias@fiocruz.br

Acompanhe os números deste periódico no seu leitor de RSS

Versão para download de PDF

PDF

Inglês

Versões e tradução automática

Versão original do texto

English

Tradução automática

Google Translator
Microsoft Translator

Como citar

RIS

BIBTEX

Outros formatos de citação e exportação:

SciELO - Scientific Electronic Library Online
Rua Dr. Diogo de Faria, 1087 – 9º andar – Vila Clementino 04037-003 São Paulo/SP - Brasil
E-mail: scielo@scielo.org

Leia a Declaração de Acesso Aberto

Métricas

Construction of three new Gateway® expression plasmids for Trypanosoma cruzi

PlumX

Mensagem

[1] + Corresponding author: eserra@fbioyf.unr.edu.ar