recombinant rep1 protein  (Jena Bioscience)

Journal: Translational Vision Science & Technology

Article Title: Expression of Rab Prenylation Pathway Genes and Relation to Disease Progression in Choroideremia

doi: 10.1167/tvst.10.8.12

Figure Lengend Snippet: Expression of genes involved in the Rab prenylation pathway. ( A ) An overview of the Rab prenylation pathway. Newly synthesized Rabs (e.g. Ras-related protein RAB27A, encoded by gene RAB27A ) are bound by the Rab Escort Proteins, REP1 (encoded by CHM ) or its paralogue REP2 (encoded by CHML ). REP proteins present Rabs to the GGTase-II heterodimer composed of α and β subunits (encoded by genes RABGGTA and RABGGTB , respectively) for prenylation. GGTase-II catalyses Rab prenylation, adding a geranylgeranyl diphosphate group to the Rab C-terminus. This allows for insertion of the Rab into the target membrane where it is activated from the guanosine diphosphate (GDP) to the guanosine triphosphate (GTP)-bound state by the guanine nucleotide exchange factor (GEF). Once activated in the target membrane, the Rab protein mediates critical functions including membrane trafficking, vesicle formation, movement, and fusion (depending on the specific Rab). Following fusion, Rab-GTP is converted back to Rab-GDP by a GTPase activating protein (GAP) and returns to the cytoplasm to bind REP (dotted line). ( B ) Expression of genes in the prenylation pathway in skin-derived fibroblasts from choroideremia patients ( n = 15) relative to controls ( n = 14). CHM expression in patients with choroideremia is significantly reduced relative to controls ( P < 0.0001). No significant differences in expression were observed between patients and controls in the genes CHML ( P = 0.99), RABGGTB ( P = 0.95), or RAB27A ( P > 0.99). ( C ) Expression of CHM plotted against CHML for patients with mutations predicted to result in non-sense mediated decay of the CHM transcript ( n = 12). No inverse correlation between the levels of CHML and CHM expression was seen to suggest any compensational upregulation of CHML (Pearson R 2 = 0.23, P = 0.11).

Article Snippet: All reactions were supplemented with fresh guanosine 5′-diphosphate (GDP, 20 mM; Merck Millipore, Watford, UK), DTT (1 mM; Thermo Fisher Scientific, Loughborough, UK), and recombinant REP1 protein (2 µM, fish His-REP1; Jena Biosciences, Jena, Germany).

Techniques: Expressing, Synthesized, Derivative Assay

Journal: Translational Vision Science & Technology

Article Title: Expression of Rab Prenylation Pathway Genes and Relation to Disease Progression in Choroideremia

doi: 10.1167/tvst.10.8.12

Figure Lengend Snippet: Quantification of REP expression and level of unprenylated Rab proteins. ( A ) Fibroblast lysates from patients with choroideremia were assessed for human REP1 expression (hREP1) and the level of unprenylated Rabs following an in vitro prenylation reaction. In the in vitro prenylation assay, unprenylated Rabs incorporate a biotin-labeled analogue of geranylgeranyl-pyrophosphate, allowing the quantification of the pool of unprenylated Rabs by the level of biotin incorporated during the reaction. Each lane represents an individual patient (see for patient details). The lane labeled with an asterisk (*) shows detection of a truncated REP1 band in the patient with an in-frame deletion of exons 2 and 3 (c.117_314del; p.R40_S105del). ( B ) Fibroblast lysates from control patients assessed for hREP1 expression and levels of unprenylated Rabs. ( C ) Fibroblast lysates from patients with choroideremia (see for patient details) and controls assessed for hREP2 expression. Detection of hREP2 requires loading of a larger quantity of protein per lane, leading to smearing of actin bands. ( D ) Quantification of hREP1 expression and the pool of unprenylated Rabs by densitometry in patients with choroideremia and normalized to expression in controls. REP1 expression in choroideremia patients is significantly reduced compared to controls ( P < 0.0001, Student's t -test). The pool of unprenylated Rabs is significantly increased in patients with choroideremia ( P < 0.001, Mann Whitney test). ( E , F ) Plot of relative quantification of REP1, unprenylated Rabs, and REP2 in patients with choroideremia relative to controls and their half-life of the degeneration of mean autofluorescence area. The rate of choroideremia progression measured by autofluorescence half-life is not correlated with REP1 expression (Pearson R 2 = 0.02, P = 0.66), the quantity of unprenylated Rabs (Spearman r = −0.4, P = 0.19), and REP2 expression (Spearman r = 0.14, P = 0.66).

Article Snippet: All reactions were supplemented with fresh guanosine 5′-diphosphate (GDP, 20 mM; Merck Millipore, Watford, UK), DTT (1 mM; Thermo Fisher Scientific, Loughborough, UK), and recombinant REP1 protein (2 µM, fish His-REP1; Jena Biosciences, Jena, Germany).

Techniques: Expressing, In Vitro, Labeling, MANN-WHITNEY