CD44 aptamer mediated cargo delivery to lysosomes of retinal pigment epithelial cells to prevent age-related macular degeneration.

Age related macular degeneration (AMD) is a progressive, neurodegenerative disorder that leads to the severe loss of central vision in elderlies. The health of retinal pigment epithelial (RPE) cells is critical for the onset of AMD. Chronic oxidative stress along with loss of lysosomal activity is a major cause for RPE cell death during AMD. Hence, development of a molecule for targeted lysosomal delivery of therapeutic protein/drugs in RPE cells is important to prevent RPE cell death during AMD. Using human RPE cell line (ARPE-19 cells) as a study model, we confirmed that hydrogen peroxide (H2O2) induced oxidative stress results in CD44 cell surface receptor overexpression in RPE cells; hence, an important target for specific delivery to RPE cells during oxidative stress. We also demonstrate that the known nucleic acid CD44 aptamer - conjugated with a fluorescent probe (FITC) - is delivered into the lysosomes of CD44 expressing ARPE-19 cells. Hence, as a proof of concept, we demonstrate that CD44 aptamer may be used for lysosomal delivery of cargo to RPE cells under oxidative stress, similar to AMD condition. Since oxidative stress may induce wet and dry AMD, both, along with proliferative vitreoretinopathy, CD44 aptamer may be applicable as a carrier for targeted lysosomal delivery of therapeutic cargoes in ocular diseases showing oxidative stress in RPE cells.

Megaplasmid - A promising tool for higher protein production.

Recombinant proteins have an increasing demand due to their application spanning across different fields. Hence, investigating strategies to increase the yield of recombinant proteins are highly significant. To achieve high yield, optimization of various parameters such as temperature, pH, aeration, inducer concentration, etc. are necessary. However, these parameters maximize the product yield of only the single open reading frame (ORF). A conventional single ORF would produce limited transcripts. Our strategy describes the generation of a tandem repeat of ORF and vector backbone, termed as megafragment (MF), followed by circularization and retaining of megaplasmid (MP) in E. coli, thereby, maximizing the protein production. We demonstrate the generation of megafragment through concatemer chain reaction and devised a method to purify megafragment from other shorter fragments. Linker was added to either end of the ORF to mediate homologous recombination and then transformed into E. coli cells to circularize the megafragment to form megaplasmid (ligase-free cloning technology). Megaplasmid can be a promising tool for higher protein expression as compared to single ORF containing plasmids. Also, E. coli BLR (DE3) and recA null strains were used here for demonstrating megaplasmid expression in the cell. The novelty of this work is the maintenance of the megaplasmid during the expression, which enables the expression of proteins at a high level.

Automation aided optimization of cloning, expression and purification of enzymes of the bacterial sialic acid catabolic and sialylation pathways enzymes for structural studies.

The process of obtaining a well-expressing, soluble and correctly folded constructs can be made easier and quicker by automating the optimization of cloning, expression and purification. While there are many semiautomated pipelines available for cloning, expression and purification, there is hardly any pipeline that involves complete automation. Here, we achieve complete automation of all the steps involved in cloning and in vivo expression screening. This is demonstrated using 18 genes involved in sialic acid catabolism and the surface sialylation pathway. Our main objective was to clone these genes into a His-tagged Gateway vector, followed by their small-scale expression optimization in vivo. The constructs that showed best soluble expression were then selected for purification studies and scaled up for crystallization studies. Our technique allowed us to quickly find conditions for producing significant quantities of soluble proteins in Escherichia coli, their large-scale purification and successful crystallization of a number of these proteins. The method can be implemented in other cases where one needs to screen a large number of constructs, clones and expression vectors for successful recombinant production of functional proteins.