Biology subjects

Mishra, P. M.

Publications and source records attributed to Mishra, P. M..

3 recordsLinked to original sources

bioRxiv2023

A broadly protective CHO cell expressed recombinant spike protein subunit based vaccine (IMT-CVAX) against SARS-CoV-2

Protective immunity induced by COVID-19 vaccines is mediated mainly by spike (S) protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Here, we report the development of a recombinant prefusion stabilized SARS-CoV-2 spike protein-subunit-based COVID-19 vaccine produced in the mammalian cell line. The gene encoding ectodomain (ECD) of the spike protein was engineered and cloned into Freedom pCHO 1.0, a mammalian expression vector, and subsequently expressed in the Chinese Hamster Ovary suspension cell line (CHO-S). The recombinant S protein ectodomain (hereafter referred to as IMT-CVAX) was purified using a combination of tangential flow filtration and liquid chromatography. Biochemical and biophysical characterization of IMT-CVAX was done to ensure its vital quality attributes. Intramuscular immunization of mice with two doses of adjuvanted IMT-CVAX elicited a strong anti-Spike IgG response. In pseudovirus-based assays, IMT-CVAX- immune mice sera exhibited a broad-spectrum neutralization of several SARS-CoV-2 variants of concern (VoCs). Golden Syrian Hamster immunized with IMT-CVAX provided excellent protection against SARS-CoV-2 infection, and, hamster immune sera neutralized the live SARS-CoV-2 virus. The adjuvanted IMT-CVAX induced robust Tfh-cells response and germinal center (GC) reaction in human ACE2 receptor-expressing transgenic mice. The findings of this study may pave the way for developing next-generation protein subunit-based vaccines to combat the existing SARS-CoV-2 and its emerging VoCs. The IMT-CVAX is produced using a scalable process and can be used for large-scale vaccine production in an industrial setup.

microbiology↗

bioRxiv2022

Investigating the folding dynamics of NS2B protein of Zika virus

NS2B protein of the Zika virus acts as a co-factor for NS3 protease where only the cytosolic domain of NS2B is sufficient for the protease activity. At the same time, NS2B also involves in remodeling the NS3 protease structure. In isolation, we previously proved the NS2B cytosolic domain (residues 49-95) as a disordered type peptide conformation. Further, this study investigated the overall dynamics of NS2B full-length protein. Our Alphafold2 structure modeling system revealed surprising similarities between selected flavivirus NS2B proteins. This similarity reflects that the NS2B protein across flavivirus is conserved fold-wise. The MD simulation of Zika virus NS2B full-length protein shows that the cytosolic domain as a part of full-length protein is a disorder region supporting our previous experimental finding, which suggests the disordered nature of the cytosolic domain in isolation. Since the cytosolic domain of NS2B is essential for protease activity, we have also investigated the folding and dynamics of the NS2B cytosolic domain (residues 49-95) that shows the disorder to alpha helix transition in TFE. On the other hand, in the presence of SDS, macromolecular crowder like ficoll and PEG do not induce secondary structural change. This dynamics study could have implications for some unknown folds of the NS2B protein.

biophysics↗

bioRxiv2021

P53 TAD2 Domain (38-61) Forms Amyloid-like Aggregates in Isolation

In many cases, when cellular machinery is unable to restore changed protein conformations, they start sticking through exposed hydrophobic patches and form aggregates. A strong association between protein aggregation and Human diseases (such as Alzheimers, Parkinsons, and Huntingtons disease) is well proven. p53 is a transcription factor that is also known as the guardian of the genome associated with cellular processes such as DNA repair, apoptosis, senescence, control of cell cycle, stress signaling and cellular homeostasis. The loss of function mutations in p53 have been implicated in several cancers. Experimental evidences have proposed a possible link between cancer and protein aggregation in evidence of the implication of amyloidogenic mutant proteins in ten different types of cancer. Aggregation studies focusing on different P53 domains, mostly, the central core domain and its mutants under the influence of various environmental conditions and P53 TAD domain (1-63) have been reported. P53 TADs interact with diverse cellular factors to modulate the function of P53 and elicit appropriate cellular response under different stress conditions. In this study, the aggregation of P53 TAD2 domain (38-61) have been studied in isolation. The aggregates were generated in-vitro in acidic pH conditions after in-silico scoring for amyloidogenic propensity and characterized using dye-based assays (ThT and bis-ANS fluorescence), CD spectroscopy, and microscopy (SEM, TEM and AFM). It was observed that P53 TAD2 follows nucleation-dependent kinetics and forms amyloid-like aggregates. On reductionists approach, this study highlights the nature of P53 TAD2 domain (amino acids 38-61) aggregation.

biochemistry↗