petm 30  (New England Biolabs)

Journal: Genes

Article Title: Functional Analyses of a Putative, Membrane-Bound, Peroxisomal Protein Import Mechanism from the Apicomplexan Protozoan Toxoplasma gondii

doi: 10.3390/genes9090434

Figure Lengend Snippet: ( A ) Toxoplasma Peroxin 5 (Pex5) contains structural characteristics conserved in yeast and human Pex5. Bioinformatic comparison of the Toxoplasma Pex5 protein sequence with yeast and human counterparts indicated the presence of critical cysteine and lysine residues (C, K), pentapeptide motifs (WxxxF/FxxxW) and structural domains (Pex7-binding region and TetratricoPeptide Repeat [TPR] domains which bind peroxisomal-targeting signal 1 [PTS1] ligands) characteristic of Pex5. Additionally, a region homologous to the Fox1 binding site in yeast Pex5 is also found in Toxoplasma Pex5; indicated by black arrowheads. ( B ) Sequence alignment of the Fox1 binding site in yeast Pex5 ( Sc Pex5 251-267 ) with Toxoplasma Pex5 ( Tg Pex5 442-456 ), arrows indicate positions in Sc Pex5 critical for Fox1 binding .

Article Snippet: The resulting pETM-30- Tg SCP2 PTS1 and pETM-30- Tg Pex5C were transformed into E. coli BL21 (DE3) (ThermoFisher Scientific), and grown in pH 7.5 buffered Luria-Bertani (LB) medium, supplemented with 1% ( w / v ) glucose and 50 mg/mL kanamycin (Sigma-Aldrich) at 37 °C.

Techniques: Sequencing, Binding Assay

Journal: Genes

Article Title: Functional Analyses of a Putative, Membrane-Bound, Peroxisomal Protein Import Mechanism from the Apicomplexan Protozoan Toxoplasma gondii

doi: 10.3390/genes9090434

Figure Lengend Snippet: Tg Pex5 complements for the loss of Pex5 in Δ pex5 mutant yeast when grown on medium containing either glucose or oleate as the sole carbon source. No complementation is indicated by a mislocalization of the heterologously expressed GFP-SKL to the cytosol as seen for the empty vector control ( A ). Functional complementation restored peroxisomal import of the GFP-SKL and led to the appearance of a punctate pattern, as seen upon expression of the gene coding for yeast Pex5 ( B ), and the gene encoding Tg Pex5 ( C ). These results indicated that Tg Pex5 can functionally replace the yeast Pex5p in PTS1-protein import into peroxisomes. Scale bar is 5 µm.

Article Snippet: The resulting pETM-30- Tg SCP2 PTS1 and pETM-30- Tg Pex5C were transformed into E. coli BL21 (DE3) (ThermoFisher Scientific), and grown in pH 7.5 buffered Luria-Bertani (LB) medium, supplemented with 1% ( w / v ) glucose and 50 mg/mL kanamycin (Sigma-Aldrich) at 37 °C.

Techniques: Mutagenesis, Plasmid Preparation, Functional Assay, Expressing

Journal: Genes

Article Title: Functional Analyses of a Putative, Membrane-Bound, Peroxisomal Protein Import Mechanism from the Apicomplexan Protozoan Toxoplasma gondii

doi: 10.3390/genes9090434

Figure Lengend Snippet: Interaction of Tg SCP2 PTS1 and Tg PEX5C in vitro. Pull-down assay was performed using the bait Tg SCP2 PTS1 with a HIS-GST tag. Eluates of the pull-down without prey protein (Lane 1, negative control) and Tg PEX5C (Lane 2) are shown. Molecular weight markers are in kilodaltons.

Article Snippet: The resulting pETM-30- Tg SCP2 PTS1 and pETM-30- Tg Pex5C were transformed into E. coli BL21 (DE3) (ThermoFisher Scientific), and grown in pH 7.5 buffered Luria-Bertani (LB) medium, supplemented with 1% ( w / v ) glucose and 50 mg/mL kanamycin (Sigma-Aldrich) at 37 °C.

Techniques: In Vitro, Pull Down Assay, Negative Control, Molecular Weight