Journal: PLoS ONE
Article Title: Computational and Functional Characterization of Angiogenin Mutations, and Correlation with Amyotrophic Lateral Sclerosis
doi: 10.1371/journal.pone.0111963
Figure Lengend Snippet: A) Coomassie stained SDS-PAGE gel showing wild-type Angiogenin-GST (lane 2), D22G (lane 3) and L35P (lane 4) proteins after Ni-NTA affinity purification. Lane 1 contains molecular weight markers. The Angiogenin proteins (14.1 kDa) are all tagged with Glutathione S-transferase (GST, ∼26 kDa) for soluble expression in E. coli . B) Plot showing CD spectra for wild-type (black), D22G (green), and L35P (brown) Angiogenin-GST proteins. Samples were diluted in PBS to yield a concentration of 0.4 mg/ml; three spectra were recorded, averaged and plotted after subtracting the buffer baseline for each sample. C) Ribonucleolytic activity of wild-type (black), D22G (green) and L35P (red) Angiogenin-GST proteins measured using yeast tRNA as substrate. The proteins, at concentrations of 0.05 to 0.5 mg/ml, were incubated with yeast tRNA (2 mg/ml) at 37°C for 2 hours. Undigested tRNA was precipitated by addition of ice-cold perchloric acid, and the absorbance of the supernatents was measured at 260 nm; data were collected from three independent experiments for each protein concentration. Student’s t -test of three independent experiments shows that the difference between wild-type and each of the three mutant protein is significant (n = 3; p<0.05). D) The loss of ribonucleolytic activity of D22G and L35P mutants compared to wild-type Angiogenin-GST. The amount of protein required to generate 1.0 optical density (OD) is compared with wild-type Angiogenin-GST to generate same OD unit for mutants.
Article Snippet: The cDNA for human ANG gene (369 bp) was amplified by PCR from the plasmid pCMV6-XL4 (OriGene) and eventually cloned in the BamHI and EcoRI restriction sites of the E. coli expression vector pGEX-6P-2 (GE-Healthcare), with a C-terminal hexa-histidine His-tag.
Techniques: Staining, SDS Page, Affinity Purification, Molecular Weight, Expressing, Concentration Assay, Activity Assay, Incubation, Protein Concentration, Mutagenesis
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