muramidase and sarkosyl

Amyloid fibril formation is responsible for several neurodegenerative diseases and are formed when native proteins misfold and stick together with different interactive forces. In the present study, we have determined the mode of interaction of the anionic surfactant sarkosyl with hen egg white lysozyme (HEWL) [EC No. 3.2.1.17] at two pHs (9.0 and 13.0) and investigated its impact on fibrillogenesis. Our data suggested that sarkosyl is promoting amyloid fibril formation in HEWL at the concentration range between 0.9 and 3.0 mM and no amyloid fibril formation was observed in the concentration range of 3.0-20.0 mM at pH 9.0. The results were confirmed by several biophysical and computational techniques, such as turbidity measurement, dynamic light scattering, Raleigh scattering, ThT fluorescence, intrinsic fluorescence, far-UV CD and atomic force microscopy. Sarkosyl was unable to induce aggregation in HEWL at pH 13.0 as confirmed by turbidity and RLS measurements. HEWL forms larger amyloid fibrils in the presence of 1.6 mM of sarkosyl. The spectroscopic, microscopic and molecular docking data suggest that the negatively charged carboxylate group and 12-carbon hydrophobic tail of sarkosyl stimulate amyloid fibril formation in HEWL via electrostatic and hydrophobic interaction. This study leads to new insight into the process of suppression of fibrillogenesis in HEWL which can be prevented by designing ligands that can retard the electrostatic and hydrophobic interaction between sarkosyl and HEWL.

Topics: Amyloid; Animals; Circular Dichroism; Dynamic Light Scattering; Fluorescence; Hydrogen-Ion Concentration; Hydrophobic and Hydrophilic Interactions; Microscopy, Atomic Force; Molecular Docking Simulation; Muramidase; Protein Aggregates; Sarcosine; Static Electricity; Surface-Active Agents

Chromatography-based protein refolding is widely used. Detergent is increasingly used for protein solubilization from inclusion bodies. Therefore, it is necessary to develop a refolding method for detergent-denatured/solubilized proteins based on liquid chromatography. In the present work, sarkosyl-denatured/dithiothreitol-reduced lysozyme was used as a model, and a refolding method based on ion exchange chromatography, assisted by β-cyclodextrin, was developed for refolding detergent-denatured proteins. Many factors affecting the refolding, such as concentration of urea, concentration of β-cyclodextrin, pH and flow rate of mobile phases, were investigated to optimize the refolding conditions for sarkosyl-denatured lysozymes. The results showed that the sarkosyl-denatured lysozyme could be successfully refolded using β-cyclodextrin-assisted ion exchange chromatography.

Topics: Animals; beta-Cyclodextrins; Chickens; Chromatography, Ion Exchange; Detergents; Hydrogen-Ion Concentration; Muramidase; Protein Denaturation; Protein Refolding; Sarcosine; Urea

We characterized a phage antibody in which an Fv fragment, namely, a free VH fragment noncovalently associated with a VL fragment that is fused with a truncated cpIII molecule (VL-DeltacpIII), is expressed on the phage surface. D1.3 antibody specific for hen egg-white lysozyme was used as a model system. Both VH and VL-DeltacpIII fragments were stably expressed and associated with each other to form a faithful antigen-binding site. The results of Western blotting indicated that more than 5% of phages expressed the Fv fragment on their surface. Analysis of the kinetics of binding of the phage antibody to the antigen suggested the possibility of presence of phages having multiple-binding sites on a single phage particle.

Topics: Animals; Antibodies, Viral; Antigen-Antibody Reactions; Bacteriophage M13; Binding Sites, Antibody; Biosensing Techniques; Chickens; Chromatography, High Pressure Liquid; Detergents; Egg White; Immunoglobulin Fragments; Muramidase; Recombinant Fusion Proteins; Sarcosine; Staphylococcal Protein A

We have developed a novel plasmid isolation procedure and have adapted it for use on an automated nucleic acid extraction instrument. The protocol is based on the finding that phenol extraction of a 1 M guanidinium thiocyanate solution at pH 4.5 efficiently removes genomic DNA from the aqueous phase, while supercoiled plasmid DNA is retained in the aqueous phase. S1 nuclease digestion of the removed genomic DNA shows that it has been denatured, which presumably confers solubility in the organic phase. The complete automated protocol for plasmid isolation involves pretreatment of bacterial cells successively with lysozyme, RNase A, and proteinase K. Following these digestions, the solution is extracted twice with a phenol/chloroform/water mixture and once with chloroform. Purified plasmid is then collected by isopropanol precipitation. The purified plasmid is essentially free of genomic DNA, RNA, and protein and is a suitable substrate for DNA sequencing and other applications requiring highly pure supercoiled plasmid.

Topics: Automation; Detergents; DNA, Bacterial; Genetic Techniques; Guanidines; Hydrogen-Ion Concentration; Muramidase; Phenols; Plasmids; RNA, Bacterial; Sarcosine; Solutions; Thiocyanates