Biology subjects

Makhija, S.

Publications and source records attributed to Makhija, S..

3 recordsLinked to original sources

bioRxiv2021

Efficient tagging and purification of endogenous proteins for structural studies by single particle cryo-EM

A major bottleneck in structural biology is producing biologically relevant samples at sufficient quantities. This is particularly true for large protein assemblies where conventional techniques of gene overexpression require substantial optimization, hampering structural studies and drug development efforts. Here we describe a method combining CRISPR/Cas gene editing and fluorescence cell sorting to rapidly tag and purify endogenous human proteins from cell lines, enabling structural analysis of native proteins that are properly folded and assembled. We applied this approach to study the human proteasome from HEK cells and rapidly determined structures of major proteasomal complexes. Structures of the PA28-20S complex reveal the native subunit stoichiometry of PA28 and a distinct functional state of the complex. The efficient strategy for tagging and extracting endogenous proteins described here will enable the structural study of many challenging targets and provide more biologically relevant samples for research and therapeutic development.

molecular biology↗

bioRxiv2020

Versatile labeling and detection ofendogenous proteins using tag-assistedsplit enzyme complementation

Recent advances in genome engineering have expanded our capabilities to study proteins in their natural states. In particular, the ease and scalability of knocking-in small peptide tags has enabled high throughput tagging and analysis of endogenous proteins. To improve enrichment capacities and expand the functionality of knock-ins using short tags, we developed the tag-assisted split enzyme complementation (TASEC) approach, which uses two orthogonal small peptide tags and their cognate binders to conditionally drive complementation of a split enzyme upon labeled protein expression. Using this approach, we have engineered and optimized the tag-assisted split HaloTag complementation system (TA-splitHalo) and demonstrated its versatile applications in improving the efficiency of knock-in cell enrichment, detection of protein-protein interaction, and isolation of biallelic gene edited cells through multiplexing.

bioengineering↗

bioRxiv2020

Faunistic study on the freshwater ciliates from Delhi, India

The ciliated protist communities show richness as well as uniqueness. This is true both for aquatic and soil ciliates. Delhi region lies in the subtropical semi-arid zone wherein the temperatures are highest in May-June and lowest in January. It also receives its monsoonal rainfall during the month of July-August. Thus, the region offers ideal conditions for the growth and proliferation of aquatic living beings. During the past three decades, a series of investigation has been carried out on the freshwater free-living ciliate fauna from fourteen sites at the river Yamuna and different freshwater bodies in Delhi. Samples were brought to the laboratory, ciliates were identified using live-cell observations and silver staining methods. A large number of Spirotrich species and a lower proportion belonging to class Heterotrichea, Litostomaea, Phyllopharyngea, Oligohymenophorea, Prostomatea, and Colpodea were identified. A total of 55 species belonging to 7 classes, 16 orders, 26 families, 40 genera were identified and documented. Ciliate diversity was found to be highest in the water sample from Okhla bird sanctuary (OBS). All ciliate species recorded during the present study have been listed and their general characteristics have been discussed.

zoology↗