Protein a quantitation by competitive ELISA using penicillinase

Summary A simple, sensitive and specific competitive ELISA method using penicillinase as a marker for quantitation of protein A has been developed. The sensitivity of the assay is 20 ng/ml. This method is useful for estimating the protein A concentration in fermented and extracted samples.     

Quantitation of protein expression in a cell-free system: Efficient detection of yields and <Superscript>19</Superscript>F NMR to identify folded protein

We have developed an efficient and novel filter assay method, involving radioactive labelling and imaging, to quantify the expression of soluble proteins from a cell-free translation system. Here this method is combined with the conformational sensitivity of ¹⁹F NMR to monitor the folded state of the expressed protein. This report describes the optimisation of 6-fluorotryptophan incorporation in a His-tagged human serum retinol-binding protein (RBP), a disulphide bonded -barrel protein. Appropriate reagent concentrations for producing fluorine labelled RBP in a cell-free translation system are described. It is shown that ¹⁹F NMR is a suitable method for monitoring the production of correctly folded protein from a high-throughput expression system.     

Simple Detection Methods for Antinutritive Factor β-ODAP Present in Lathyrus sativus L. by High Pressure Liquid Chromatography and Thin Layer Chromatography

Lathyrus sativus L. (Grass pea) is the source for cheap and nutritious food choice in drought and famine susceptible zones in greater part of North India and Africa. The non-protein amino acid β-N-oxalyl-L-α,β-diaminopropionic acid (β-ODAP) has been known for decades for its potent neurotoxic effect, causing irreversible neurodegenerative disease “neurolathyrism”, present in both seed and leaf of Lathyrus sativus L. and other species in varying proportions. It is crucial to establish a rapid as well as reliable detection methodology for β-ODAP content in various Lathyrus plants. Currently available HPLC based methods involve multi-step derivatization of the sample. To overcome this, we have developed β-ODAP analysis method by HPLC without any prior derivatization. This method is statistically significant in the range of 2 to 100μg/ml and exhibited linear response with r ² > 0.99. Limit of detection and quantitation of the later method was determined to be 5.56 μg/ml and 16.86 μg/ml, respectively. In addition to this, a TLC based method has also been developed. The limit of detection of β-ODAP is 0.6μg and for its substrate, L-1,2-diaminopropionic acid is 5μg. Both HPLC and TLC methods were validated by conducting in-vitro bioconversion test to detect the presence of biocatalyst in plant extract. This method is economical, rapid and simple.     

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.     

Application of aptamers in diagnostics,drug-delivery and imaging

Aptamers are small, single-stranded oligonucleotides (DNA or RNA) that bind to their target with high specificity and affinity. Although aptamers are analogous to antibodies for a wide range of target recognition and variety of applications, they have significant advantages over antibodies. Since aptamers have recently emerged as a class of biomolecules with an application in a wide array of fields, we need to summarize the latest developments herein. In this review we will discuss about the latest developments in using aptamers in diagnostics, drug delivery and imaging. We begin with diagnostics, discussing the application of aptamers for the detection of infective agents itself, antigens/toxins (bacteria), biomarkers (cancer), or a combination. The ease of conjugation and labelling of aptamers makes them a potential tool for diagnostics. Also, due to the reduced off-target effects of aptamers, their use as a potential drug delivery tool is emerging rapidly. Hence, we discuss their use in targeted delivery in conjugation with siRNAs, nanoparticles, liposomes, drugs and antibodies. Finally, we discuss about the conjugation strategies applicable for RNA and DNA aptamers for imaging. Their stability and self-assembly after heating makes them superior over protein-based binding molecules in terms of labelling and conjugation strategies.     

Effect of 17α-methyl testosterone incorporated diets on growth of spotted snakehead,Channa punctatus and white carp,Cirrhinus mrigala

The effect of different concentrations of 17α-methyl testosterone incorporated diet on growth performance in the fry of Channa punctatus and Cirrhinus mrigala was evaluated. Four different doses of hormone such as 60, 80, 100 and 120?mg/kg in C. punctatus and 40, 60, 80 and 100?mg/kg in C. mrigala were administered through diet for a period of 90?days.Fifth group on a hormone free diet served as a control. The growth performance in terms of length and weight gain of the fry receiving 100?mg/kg in C. punctatus and 60?mg/kg in C. mrigala were significantly higher than those receiving 80, 120 and 0 (untreated control) mg hormone per kg feed. The highest specific growth rate (0.864?±?1.18%WG d^?1) at 100?mg/Kg diet and (2.47?±?1.26%WG d^?1) at 60?mg/kg diet were observed in C. punctatus and C. mrigala respectively, showing positive influence of hormone incorporated diet on the growth performance. However, the survival rate of both the species remained unaffected by different dosages of 17α-methyl testosterone.     

Tailor-Made Ezrin Actin Binding Domain to Probe Its Interaction with Actin In-Vitro

Ezrin, a member of the ERM (Ezrin/Radixin/Moesin) protein family, is an Actin-plasma membrane linker protein mediating cellular integrity and function. In-vivo study of such interactions is a complex task due to the presence of a large number of endogenous binding partners for both Ezrin and Actin. Further, C-terminal actin binding capacity of the full length Ezrin is naturally shielded by its N-terminal, and only rendered active in the presence of Phosphatidylinositol bisphosphate (PIP2) or phosphorylation at the C-terminal threonine. Here, we demonstrate a strategy for the design, expression and purification of constructs, combining the Ezrin C-terminal actin binding domain, with functional elements such as fusion tags and fluorescence tags to facilitate purification and fluorescence microscopy based studies. For the first time, internal His tag was employed for purification of Ezrin actin binding domain based on in-silico modeling. The functionality (Ezrin-actin interaction) of these constructs was successfully demonstrated by using Total Internal Reflection Fluorescence Microscopy. This design can be extended to other members of the ERM family as well.     

Soni-removal of nucleic acids from inclusion bodies

Inclusion bodies (IBs) are commonly formed in Escherichiacoli due to over expression of recombinant proteins in non-native state. Isolation, denaturation and refolding of these IBs is generally performed to obtain functional protein. However, during this process IBs tend to form non-specific interactions with sheared nucleic acids from the genome, thus getting carried over into downstream processes. This may hinder the refolding of IBs into their native state. To circumvent this, we demonstrate a methodology termed soni-removal which involves disruption of nucleic acid–inclusion body interaction using sonication; followed by solvent based separation. As opposed to conventional techniques that use enzymes and column-based separations, soni-removal is a cost effective alternative for complete elimination of buried and/or strongly bound short nucleic acid contaminants from IBs.