Journal: International Journal of Molecular Medicine
Article Title: Ribonuclease T2 from Aspergillus fumigatus promotes T helper type 2 responses through M2 polarization of macrophages
Figure Lengend Snippet: Expression, purification, and RNase activity of r Af RNASET2. (A) Analysis of the prokaryotic recombinant expression vector pET-His- Af RNASET2 (without signal peptides, Glutamic acid 26-Arginine 407) by double enzyme digestion. Lane M indicates the DNA marker. The 1,200 bp band indicates the Af RNASET2 cDNA insert. (B) SDS-PAGE analysis of pET-His-Af RNASET2 expression. Lane M, protein marker; lane 1, E. coli transformed with pET-His-Af RNASET2 prior to IPTG induction; lane 2, E. coli transformed with pET-His- Af RNASET2 following IPTG induction; lane 3, sediment from E. coli transformed with pET-His- Af RNASET2 plasmid following IPTG induction, and ultrasonication. (C) SDS-PAGE of renatured r Af RNASET2 protein following purification using Ni-NTA affinity chromatography. Lane M indicates the protein marker. (D) Analysis of RNase activity of r Af RNASET2 (1 µ g) with yeast RNA (50 µ g) at 37°C at 20, 40, 60, 80 and 100 min). (E) Analysis of RNase activity of r Af RNASET2 with or without RNasin (RNAse inhibitor) pretreatment. RNase enzymolysis products were analyzed using 1% agarose gel electrophoresis. E. coli, Escherichia coli ; r, recombinant; Af, Aspergillus fumigatus ; RNASET2, T2 ribonuclease; IPTG, isopropyl-β-d-thiogalactopyranoside.
Article Snippet: Expression system components and reagents The following reagents were purchased for recombinant protein expression: Escherichia coli (E. coli ) BL21 (DE3) plysS cells (Merck KGaA), pMD 19-T vectors (Takara Biotechnology Co., Ltd.), pET-His vectors (Wuhan Miaoling Bioscience & Technology Co., Ltd.), and primer STAR HS DNA polymerase (Takara Biotechnology Co., Ltd.).
Techniques: Expressing, Purification, Activity Assay, Recombinant, Plasmid Preparation, Positron Emission Tomography, Marker, SDS Page, Transformation Assay, Affinity Chromatography, Agarose Gel Electrophoresis