Studies on pH-Controlled Transition of Myoglobin Capsules from Hollow to Multilayered Structures

In this study, myoglobin biocapsules (lyophilisomes) of various types and sizes were prepared using a combination of freezing—annealing and immobilization. Hollow lyophilisomes of pure myoglobin below and above its isoelectric point were designed and changes in their secondary structure based on variations in pH were studied by circular dichroism spectroscopy and confocal laser scanning microscopy. The study results show that permeability and size of these capsules change as pH changes; besides, near the isoelectric point, large swollen multiwalled capsules are formed, which when exposed to buffers of lower pH exhibited a tendency to reverse to hollow structures. Adsorption of these capsules onto solid surfaces has been studied using quartz crystal microbalance and contact angle measurements. Thus, pH shift may be a useful post-preparation approach for stimuli-responsive modification of the surface roughness, porosity and permeability of pre-assembled polypeptide microcapsules

Influence of Ficoll on urea induced denaturation of fibrinogen

Ficoll is a neutral, highly branched polymer used as a molecular crowder in the study of proteins. Ficoll is also part of Ficoll-Paque used in biology laboratories to separate blood to its components (erythrocytes, leukocytes etc.,). Role of Ficoll in the urea induced denaturation of protein Fibrinogen (Fg) has been analyzed using fluorescence, circular dichroism, molecular docking and interfacial studies. Fluorescence studies show that Ficoll prevents quenching of Fg in the presence of urea. From the circular dichroism spectra, Fg shows conformational transition to random coil with urea of 6 M concentration. Ficoll helps to shift this denaturation concentration to 8 M and thus constraints by shielding Fg during the process. Molecular docking studies indicate that Ficoll interacts favorably with the protein than urea. The surface tension and shear viscosity analysis shows clearly that the protein is shielded by Ficoll

Molecular evolution of single chain fragment variable (scFv) for diagnosis of lymphatic filariasis

Endemic countries with lymphatic filariasis are striving towards the Global Program to Eliminate Lymphatic Filariasis (GPELF) by 2020. Efficient and cost-effective diagnostic tools to assess active filarial infection are critical to eradicate lymphatic filariasis. Detection of circulating filarial antigens in sera is one of the precise methods to identify this infection. Monoclonal antibodies and single chain fragment variable (scFv) against Wuchereria bancrofti antigen SXP1 have been developed for antigen detection. Molecular cloning of scFv for recombinant expression has laid a platform for developing novel genetic constructs with enhanced reactivity. In this study, a simple procedure is developed to create diverse libraries of scFv based on a single DNA framework with all the requisites for an in vitro protein synthesis and ribosomal display. Error Prone-PCR was performed to incorporate random mutations and screened by ribosome display technique to isolate evolved scFv. Evolved scFv with six mutations showed tenfold increase in affinity compared to wild-type scFv for rWbSXP1. In silico studies showed that four mutations introduced unique molecular interactions between the evolved scFv and SXP1. Reactivity with asserted clinical samples of endemic normals (EN), microfilariaemic (MF), chronic pathology (CP) and non-endemic normals (NEN) showed significant augment (59.69%, p < 0.0001) in reactivity to MF samples with evolved scFv in comparison to wild-type scFv. Sensitivity of scFv was increased from 15.62 ng to 195 pg by evolved scFv in serum samples. This evolutionary method coupled with ribosome display has facilitated us to improve the reactivity of the ScFv without diminishing the specificity