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Construction of three new Gateway® expression plasmids for Trypanosoma cruzi.

Alonso VL, Ritagliati C, Cribb P, Serra EC - Mem. Inst. Oswaldo Cruz (2014)

Bottom Line: 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).

View Article: PubMed Central - PubMed

Affiliation: Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario, Rosario, Argentina.

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 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).

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: pTEX and pTREX-TAPtag-GW experimental validation (A) Coomassie and westernblot analysis of total lysates from CL Brener wild type cells (Lane 1) and thosetransfected (2) with pTEX-TAPtag-GW-GFP or pTREX-TAPtag-GW-GFP. Anti-greenfluorescent protein (GFP) (a-GFP) and anti-protein A(a-protein A) antibodies were used. Bound antibodies weredetected 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 andpTREX-TAPtag-GW-GFP. Parasites were adhered to poly-L-lysine coated slides andfixed with 4% paraformaldehyde in phosphate-buffered saline (PBS), permeabilisedwith 0.1% Triton X-100 in PBS and then incubated with a-protein Aantibodies in 1% bovine serum albumin-PBS for 3 h. Then, the slides were incubatedwith anti-mouse IgG antibody conjugated to Alexa Fluor 555 secondary antibodiesfor 1 h. The slides were mounted with VectaShield (Vector Laboratories) in thepresence of 2 µg mL-1 4’-6-diamidino-2-phenylindole (DAPI) in PBS. Images wereacquired with a Nikon Eclipse TE-2000-E2 confocal microscope. Adobe Photoshop CSv.8.0.1 (Adobe System Incorporated) and Nikon EZ-C1 FreeViewer v.3.70 (NikonCorporation) software were used to analyse the images.
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f02: : pTEX and pTREX-TAPtag-GW experimental validation (A) Coomassie and westernblot analysis of total lysates from CL Brener wild type cells (Lane 1) and thosetransfected (2) with pTEX-TAPtag-GW-GFP or pTREX-TAPtag-GW-GFP. Anti-greenfluorescent protein (GFP) (a-GFP) and anti-protein A(a-protein A) antibodies were used. Bound antibodies weredetected 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 andpTREX-TAPtag-GW-GFP. Parasites were adhered to poly-L-lysine coated slides andfixed with 4% paraformaldehyde in phosphate-buffered saline (PBS), permeabilisedwith 0.1% Triton X-100 in PBS and then incubated with a-protein Aantibodies in 1% bovine serum albumin-PBS for 3 h. Then, the slides were incubatedwith anti-mouse IgG antibody conjugated to Alexa Fluor 555 secondary antibodiesfor 1 h. The slides were mounted with VectaShield (Vector Laboratories) in thepresence of 2 µg mL-1 4’-6-diamidino-2-phenylindole (DAPI) in PBS. Images wereacquired with a Nikon Eclipse TE-2000-E2 confocal microscope. Adobe Photoshop CSv.8.0.1 (Adobe System Incorporated) and Nikon EZ-C1 FreeViewer v.3.70 (NikonCorporation) software were used to analyse the images.

Mentions: Once the lines were selected with G418, we monitored GFP expression by western blotting andfluorescence microscopy (Fig. 2). Immunoblot analysiswith commercial anti-GFP (Santa Cruz Biotechnology) and anti-Protein A (Sigma) antibodiesshowed a single band of ~60 kDa, which corresponds to the full fused protein that ispresent only in transfected parasites, but not in the wild type. No partially synthesisedor proteolytic products were observed (Fig. 2A).Immunofluorescence microscopy of transfected epimastigotes demonstrated expression of GFPand the TAP tag, which was detected with mouse anti-Protein A antibodies and Alexa Fluor555 anti-mouse (Invitrogen) as a secondary antibody. We observed a positive signal for PAonly in parasites that expressed GFP, which have a similar fluorescence pattern in thewhole cell body, but do not completely co-localise. This difference is likely because thered signal is indirect and comes from the antibodies and the GFP signal is more intense(Fig. 2B).


Construction of three new Gateway® expression plasmids for Trypanosoma cruzi.

Alonso VL, Ritagliati C, Cribb P, Serra EC - Mem. Inst. Oswaldo Cruz (2014)

: pTEX and pTREX-TAPtag-GW experimental validation (A) Coomassie and westernblot analysis of total lysates from CL Brener wild type cells (Lane 1) and thosetransfected (2) with pTEX-TAPtag-GW-GFP or pTREX-TAPtag-GW-GFP. Anti-greenfluorescent protein (GFP) (a-GFP) and anti-protein A(a-protein A) antibodies were used. Bound antibodies weredetected 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 andpTREX-TAPtag-GW-GFP. Parasites were adhered to poly-L-lysine coated slides andfixed with 4% paraformaldehyde in phosphate-buffered saline (PBS), permeabilisedwith 0.1% Triton X-100 in PBS and then incubated with a-protein Aantibodies in 1% bovine serum albumin-PBS for 3 h. Then, the slides were incubatedwith anti-mouse IgG antibody conjugated to Alexa Fluor 555 secondary antibodiesfor 1 h. The slides were mounted with VectaShield (Vector Laboratories) in thepresence of 2 µg mL-1 4’-6-diamidino-2-phenylindole (DAPI) in PBS. Images wereacquired with a Nikon Eclipse TE-2000-E2 confocal microscope. Adobe Photoshop CSv.8.0.1 (Adobe System Incorporated) and Nikon EZ-C1 FreeViewer v.3.70 (NikonCorporation) software were used to analyse the images.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4325611&req=5

f02: : pTEX and pTREX-TAPtag-GW experimental validation (A) Coomassie and westernblot analysis of total lysates from CL Brener wild type cells (Lane 1) and thosetransfected (2) with pTEX-TAPtag-GW-GFP or pTREX-TAPtag-GW-GFP. Anti-greenfluorescent protein (GFP) (a-GFP) and anti-protein A(a-protein A) antibodies were used. Bound antibodies weredetected 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 andpTREX-TAPtag-GW-GFP. Parasites were adhered to poly-L-lysine coated slides andfixed with 4% paraformaldehyde in phosphate-buffered saline (PBS), permeabilisedwith 0.1% Triton X-100 in PBS and then incubated with a-protein Aantibodies in 1% bovine serum albumin-PBS for 3 h. Then, the slides were incubatedwith anti-mouse IgG antibody conjugated to Alexa Fluor 555 secondary antibodiesfor 1 h. The slides were mounted with VectaShield (Vector Laboratories) in thepresence of 2 µg mL-1 4’-6-diamidino-2-phenylindole (DAPI) in PBS. Images wereacquired with a Nikon Eclipse TE-2000-E2 confocal microscope. Adobe Photoshop CSv.8.0.1 (Adobe System Incorporated) and Nikon EZ-C1 FreeViewer v.3.70 (NikonCorporation) software were used to analyse the images.
Mentions: Once the lines were selected with G418, we monitored GFP expression by western blotting andfluorescence microscopy (Fig. 2). Immunoblot analysiswith commercial anti-GFP (Santa Cruz Biotechnology) and anti-Protein A (Sigma) antibodiesshowed a single band of ~60 kDa, which corresponds to the full fused protein that ispresent only in transfected parasites, but not in the wild type. No partially synthesisedor proteolytic products were observed (Fig. 2A).Immunofluorescence microscopy of transfected epimastigotes demonstrated expression of GFPand the TAP tag, which was detected with mouse anti-Protein A antibodies and Alexa Fluor555 anti-mouse (Invitrogen) as a secondary antibody. We observed a positive signal for PAonly in parasites that expressed GFP, which have a similar fluorescence pattern in thewhole cell body, but do not completely co-localise. This difference is likely because thered signal is indirect and comes from the antibodies and the GFP signal is more intense(Fig. 2B).

Bottom Line: 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).

View Article: PubMed Central - PubMed

Affiliation: Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario, Rosario, Argentina.

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 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).

Show MeSH
Related in: MedlinePlus