Abstract
Metal nanomaterials are shown to enhance gene expression for rice α‐galactosidase gene (α‐Gal) in yeast cells. Au and Ag nanoparticles and their nanocomposites, silica‐Au and silica‐Ag, were prepared and characterized by UV‐vis spectroscopy and TEM technique. The rice α‐galactosidase gene was cloned into the yeast chromosome, where the cloned cells were precultured and induced into a medium containing each of the testing nanomaterials. The nanomaterials were observed to incorporate inside the cells, and no cell death has been detected during the course of gene expression. The enzyme activity was determined by a synthetic substrate, p‐nitrophenyl‐α‐D‐galctopyranoside, and the yellow product yield was recorded in a spectrophotometer at 400 nm. When Au and Ag nanoparticles were incorporated with the culture, a 3–5 fold enhancement in α‐galactosidase was observed for intracellular activity as well as the secreted activity into the medium. The secreted protein was analyzed to have a pure form and displayed as a single protein band in the SDS‐gel electrophoresis. The effects of size and chemical nature of nanomaterials on gene expression for the rice α‐galactosidase gene in yeast cells are discussed.
Highlights
The size and surface area of the nanoparticles together with their available functional groups and charges are crucial factors in targeting and the attachment of cell-specific ligands that can lead to an increased selectivity in delivery and expression of genes
We describe the use of oligonucleotide-loaded nanoparticles to enhance the expression of rice α-galactosidase gene in yeast cells
Rice α-galactosidase gene was cloned in pPIC-9k plasmid (Invitrogen, Calif, USA) and transformed into SMD 1168 yeast strain chromosomal DNA by electroporation according to the Invitrogen protocol described in EasySelect and given by Higgins and Cregg [12]
Summary
The size and surface area of the nanoparticles together with their available functional groups and charges are crucial factors in targeting and the attachment of cell-specific ligands that can lead to an increased selectivity in delivery and expression of genes. Gold and silica nanoparticles have been employed to investigate gene expression from the unamplified total human RNA [1] and in vivo in the brain [2]. It has been known for a long time that the Indian eats silver powder and the Chinese mixes gold thin films in food. Gold nanoparticles were loaded and modified with oligonucleotide and employed as the intracellular gene regulation agents for controlling protein expression in cells [3]. We describe the use of oligonucleotide-loaded nanoparticles to enhance the expression of rice α-galactosidase gene in yeast cells. Journal of Nanomaterials into medium? If the secreted activity is highly specific, will the protein expressed be a pure form and no further protein purification should be required?
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