@article {507, title = {Tailor-made ezrin actin binding domain to probe its interaction with actin in-vitro. [Protein Technology Core]}, journal = {PLoS One}, volume = {10}, year = {2015}, month = {2015}, pages = {e0123428}, abstract = {

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.

}, keywords = {Actins, Animals, Avian Proteins, Chickens, Cytoskeletal Proteins, In Vitro Techniques, Microfilament Proteins, Models, Molecular, Protein Interaction Domains and Motifs, Recombinant Fusion Proteins}, issn = {1932-6203}, doi = {10.1371/journal.pone.0123428}, author = {Shrivastava, Rohini and K{\"o}ster, Darius and Kalme, Sheetal and Mayor, Satyajit and Neerathilingam, Muniasamy} } @article {504, title = {Thioaptamers targeting dengue virus type-2 envelope protein domain III. [Protein Technology Core]}, journal = {Biochem Biophys Res Commun}, volume = {453}, year = {2014}, month = {2014 Oct 24}, pages = {309-15}, abstract = {

Thioaptamers targeting the dengue-2 virus (DENV-2) envelope protein domain III (EDIII) were developed. EDIII, which contains epitopes for binding neutralizing antibodies, is the putative host-receptor binding domain and is thus an attractive target for development of vaccines, anti-viral therapeutic and diagnostic agents. Thioaptamer DENTA-1 bound to DENV-2 EDIII adjacent to a known neutralizing antibody binding site with a dissociation constant of 154nM.

}, keywords = {Antibodies, Neutralizing, Antiviral Agents, Aptamers, Nucleotide, Base Sequence, Dengue Virus, Magnetic Resonance Spectroscopy, Viral Envelope Proteins}, issn = {1090-2104}, doi = {10.1016/j.bbrc.2014.09.053}, author = {Gandham, Sai Hari A and Volk, David E and Lokesh, Ganesh L R and Neerathilingam, Muniasamy and Gorenstein, David G} } @article {1009, title = {Tender coconut water an economical growth medium for the production of recombinant proteins in Escherichia coli. [Protein Technology Facility]}, journal = {BMC Biotechnol}, volume = {13}, year = {2013}, month = {2013 Sep 02}, pages = {70}, abstract = {

BACKGROUND: Escherichia coli is most widely used prokaryotic expression system for the production of recombinant proteins. Several strategies have been employed for expressing recombinant proteins in E.coli. This includes the development of novel host systems, expression vectors and cost effective media. In this study, we exploit tender coconut water (TCW) as a natural and cheaper growth medium for E.coli and Pichia pastoris.

RESULT: E.coli and P.pastoris were cultivated in TCW and the growth rate was monitored by measuring optical density at 600 nm (OD(600nm)), where 1.55 for E.coli and 8.7 for P.pastoris was obtained after 12 and 60 hours, respectively. However, variation in growth rate was observed among TCW when collected from different localities (0.15-2.5 at OD(600nm)), which is attributed to the varying chemical profile among samples. In this regard, we attempted the supplementation of TCW with different carbon and nitrogen sources to attain consistency in growth rate. Here, supplementation of TCW with 25 mM ammonium sulphate (TCW-S) was noted efficient for the normalization of inconsistency, which further increased the biomass of E.coli by 2 to 10 folds, and 1.5 to 2 fold in P.pastoris. These results indicate that nitrogen source is the major limiting factor for growth. This was supported by total nitrogen and carbon estimation where, nitrogen varies from 20 to 60 mg/100 ml while carbohydrates showed no considerable variation (2.32 to 3.96 g/100 ml). In this study, we also employed TCW as an expression media for recombinant proteins by demonstrating successful expression of maltose binding protein (MBP), MBP-TEV protease fusion and a photo switchable fluorescent protein (mEos2) using TCW and the expression level was found to be equivalent to Luria Broth (LB).

CONCLUSION: This study highlights the possible application of TCW-S as a media for cultivation of a variety of microorganisms and recombinant protein expression.

}, keywords = {Ammonium Sulfate, Biomass, Carbohydrates, Carbon, Cocos, Culture Media, Escherichia coli, Gene Expression, Nitrogen, Pichia, Recombinant Proteins}, issn = {1472-6750}, doi = {10.1186/1472-6750-13-70}, author = {Sekar, Narendrakumar and Veetil, Soumya Kariyadan and Neerathilingam, Muniasamy} }