ABSTRACT
To characterise the trypanosomatid-exclusive RNA-binding protein TcRBP19, we analysed the phenotypic changes caused by its overexpression. Although no evident changes were observed when TcRBP19 was ectopically expressed in epimastigotes, the metacyclogenesis process was affected. Notably, TcRBP19 overexpression also led to a decrease in the number of infected mammalian cells. These findings suggest that TcRBP19 may be involved in the life cycle progression of the Trypanosoma cruzi parasite.
Subject(s)
Animals , Protozoan Proteins/physiology , RNA-Binding Proteins/genetics , Trypanosoma cruzi/genetics , Gene Expression Regulation , Life Cycle Stages , RNA Processing, Post-Transcriptional/genetics , RNA-Binding Proteins/chemistry , RNA-Binding Proteins/metabolism , RNA-Binding Proteins/physiology , Trypanosoma cruzi/growth & development , Trypanosoma cruzi/metabolismABSTRACT
Aptamers are synthetic, relatively short (e.g., 20-80 bases) RNA or ssDNA oligonucleotides that can bind targets with high affinity and specificity, similar to antibodies, because they can fold into unique, three-dimensional shapes. For use in various assays and experiments, aptamers have been conjugated with biotin or digoxigenin to form complexes with avidin or anti-digoxigenin antibodies, respectively. In this study, we developed a method to label the 5' ends of aptamers with cotinine, which allows formation of a stable complex with anti-cotinine antibodies for the purpose of providing another affinity unit for the application in biological assays using aptamers. To demonstrate the functionality of this affinity unit in biological assays, we utilized two well-known aptamers: AS1411, which binds nucleolin, and pegaptanib, which binds vascular endothelial growth factor. Cotinine-conjugated AS1411/anti-cotinine antibody complexes were successfully applied to immunoblot, immunoprecipitation, and flow cytometric analyses, and cotinine-conjugated pegaptanib/anti-cotinine antibody complexes were used successfully in enzyme immunoassays. Our results show that cotinine-conjugated aptamer/anti-cotinine antibody complexes are an effective alternative and complementary technique for aptamer use in multiple assays and experiments.
Subject(s)
Animals , Humans , Mice , Antibodies, Anti-Idiotypic/immunology , Aptamers, Nucleotide/chemistry , Biological Assay , Cotinine/administration & dosage , Flow Cytometry , Hep G2 Cells , NIH 3T3 Cells , Phosphoproteins/chemistry , Protein Binding , RNA-Binding Proteins/chemistry , Vascular Endothelial Growth Factor A/chemistryABSTRACT
The viral genome-linked protein (VPg) of Potyviruses is covalently attached to the 5’ end of the genomic RNA. Towards biophysical characterization, the VPg coding region of Cardamom mosaic virus (CdMV) was amplified from the cDNA and expressed in E. coli. Most of the expressed VPg aggregated as inclusion bodies that were solubilized with urea and refolded with L-arginine hydrochloride. The various forms of CdMV VPg (native, denatured and refolded) were purified and the conformational variations between these forms were observed with fluorescence spectroscopy. Native and refolded CdMV VPg showed unordered secondary structure in the circular dichroism (CD) spectrum. The model of CdMV VPg was built based on the crystal structure of phosphotriesterase (from Pseudomonas diminuta), which had the maximum sequence homology with VPg to identify the arrangement of conserved amino acids in the protein to study the functional diversity of VPg. This is the first report on the VPg of CdMV, which is classified as a new member of the Macluravirus genus of the Potyviridae family.
Subject(s)
Circular Dichroism , Elettaria/metabolism , Genome, Viral/genetics , Inclusion Bodies/genetics , Inclusion Bodies/metabolism , Models, Molecular , Mosaic Viruses/genetics , Mosaic Viruses/metabolism , Plant Viruses/genetics , Plant Viruses/metabolism , Potyvirus/genetics , Potyvirus/metabolism , Protein Refolding , Protein Structure, Secondary , RNA-Binding Proteins/chemistry , RNA-Binding Proteins/genetics , RNA-Binding Proteins/isolation & purification , RNA-Binding Proteins/metabolism , Viral Proteins/chemistry , Viral Proteins/genetics , Viral Proteins/metabolismABSTRACT
A poly(A)-binding protein (PABP) with mol wt 29,000 has been purified from chickpea (Cicer arietinum) epicotyl by ammonium sulfate fractionation and Cibacron blue F3-GA chromatography, making a complex with poly(A) and elution of PABP-poly(A) complex at 45 degrees C from oligo d(T)-cellulose. The elution pattern and binding properties show that the purified protein is different from the PABP (mol. wt 72,000) reported earlier from our laboratory.
Subject(s)
Cicer/chemistry , Molecular Weight , Plant Proteins/chemistry , Plants, Medicinal , Poly(A)-Binding Proteins , RNA-Binding Proteins/chemistryABSTRACT
A poly(A)-binding protein (PABP) with mol wt 72,000 has been purified from chickpea (Cicer arietinum) epicotyls by ammonium sulfate fractionation, Cibacron blue F3-GA and poly(A) agarose chromatography. The binding properties and the specificity of binding show that the purified protein is an analogue of PABPs in other eukaryotes. This PABP is highly susceptible to proteolysis and upon degradation forms a polypeptide fragment of mol wt 21,000 which has an independent poly(A) binding activity.