Expression and functional identification of recombinant SARS-CoV-2 receptor binding domain (RBD) from E. coli system.

The receptor binding domain (RBD) of SARS-CoV-2 is located in the C-terminal of S1 subunit of the spike (S) protein which is responsible for recognizing and binding to the angiotensin-converting enzyme 2 (ACE2) receptor. The DNA encoding the SARS-CoV-2 RBD was inserted into pET-28a (+) to construct expression plasmid pET-28a (+)/RBD. The desired RBD protein was produced in E. coli Rosetta (DE) and purified by a Ni-NTA column. The recombinant RBD was analyzed by SDS-PAGE and Western blot. The flow cytometry analysis indicated that the recombinant RBD is capable of binding to human ACE2 (hACE2) in the ACE2-overexpressed HEK293A-hACE2 cells. Our results demonstrated that recombinant RBD expressed in E. coli Rosetta (DE) strain has bioactivities and can be used as an antigen for diagnosis and as a tool for the development of novel anti-viral drugs against SASR-CoV-2.

Virulent Drexlervirial Bacteriophage MSK, Morphological and Genome Resemblance With Rtp Bacteriophage Inhibits the Multidrug-Resistant Bacteria.

Phage-host interactions are likely to have the most critical aspect of phage biology. Phages are the most abundant and ubiquitous infectious acellular entities in the biosphere, where their presence remains elusive. Here, the novel Escherichia coli lytic bacteriophage, named MSK, was isolated from the lysed culture of E. coli C (phix174 host). The genome of phage MSK was sequenced, comprising 45,053 bp with 44.8% G + C composition. In total, 73 open reading frames (ORFs) were predicted, out of which 24 showed a close homology with known functional proteins, including one tRNA-arg; however, the other 49 proteins with no proven function in the genome database were called hypothetical. Electron Microscopy and genome characterization have revealed that MSK phage has a rosette-like tail tip. There were, in total, 46 ORFs which were homologous to the Rtp genome. Among these ORFs, the tail fiber protein with a locus tag of MSK_000019 was homologous to Rtp 43 protein, which determines the host specificity. The other protein, MSK_000046, encodes lipoprotein (cor gene); that protein resembles Rtp 45, responsible for preventing adsorption during cell lysis. Thirteen MSK structural proteins were identified by SDS-PAGE analysis. Out of these, 12 were vital structural proteins, and one was a hypothetical protein. Among these, the protein terminase large (MSK_000072) subunit, which may be involved in DNA packaging and proposed packaging strategy of MSK bacteriophage genome, takes place through headful packaging using the pac-sites. Biosafety assessment of highly stable phage MSK genome analysis has revealed that the phage did not possess virulence genes, which indicates proper phage therapy. MSK phage potentially could be used to inhibit the multidrug-resistant bacteria, including AMP, TCN, and Colistin. Further, a comparative genome and lifestyle study of MSK phage confirmed the highest similarity level (87.18% ANI). These findings suggest it to be a new lytic isolated phage species. Finally, Blast and phylogenetic analysis of the large terminase subunit and tail fiber protein put it in Rtp viruses' genus of family Drexlerviridae.

An efficient system to generate truncated human angiotensin converting enzyme 2 (hACE2) capable of binding RBD and spike protein of SARS-CoV2.

Human angiotensin converting enzyme 2 (hACE2) mediates the cell entry of both SARS-CoV and SARS-CoV2 and can be used as a drug target. The DNA encoding the truncated hACE2 (30-356aa) was cloned into pET-28a (+) and expressed in Escherichia coli Rosetta (DE3). The recombinant hACE2 (rhACE2) was purified by affinity chromatography on a Ni-NTA column and characterized with SDS-PAGE and Western blot. The binding activity of rhACE2 to Spike protein of SARS-CoV2 was evaluated in S protein-overexpressed HEK293A cells (HEK293A-SP cells) through flow cytometry. The prokaryotic expression system is able to produce approximately 75 mg protein per liter, which would be useful for infection mechanism study, and drug screening and development of SARS-CoV2.

Dynamics of Endocytosis and Degradation of Antibody-Drug Conjugate T-DM1 in HER2 Positive Cancer Cells.

PURPOSE: T-DM1 is an antibody-drug conjugate (ADC) consisting of trastuzumab and DM1 linked together. T-DM1 binds to human epidermal growth factor receptor-2 (HER2) in tumors and then triggers the endocytosis of T-DM1 and release of payload. Therefore, endocytosis efficacy is considered as a critical step for the initiation of T-DM1 therapy; however, the endocytosis mechanism of T-DM1 remains poorly understood. Meanwhile, HER2 is regarded as an internalization-resistant receptor, which hinders the endocytosis and effectiveness of T-DM1. The present study is to explore the T-DM1 endocytosis pathway, which may provide insights into the internalization mechanism of ADCs and help to improve efficacy. METHODS: Confocal microscopy and flow cytometry were used to analyse T-DM1 intracellular trafficking and endocytosis efficiency, while Western blot assay was performed to detect T-DM1 degradation. RESULTS: We found that intracellular T-DM1 was increased to 50% within 12 h. T-DM1 was colocalized with cholera toxin B (CTxB), a lipid raft marker, within 2 h and then degraded in lysosome. Upon overexpression of caveolin-1 (CAV-1) and utilization of caveolae/lipid-raft disruptors, we found that temporal CAV-1 upregulation significantly facilitated T-DM1 endocytosis and degradation, whereas nystatin and lovastatin disrupted caveolae/lipid-raft structure and inhibited T-DM1 degradation. We demonstrate that T-DM1 internalizes through the lipid raft-mediated endocytosis in a CAV-1 dependent manner, rather than through the clathrin-mediated endocytosis in HER2-positive cancer cells. CONCLUSION: Our findings suggest that modulation of the caveolae/lipid-raft mediated endocytosis may be a possible option for improving the clinical therapeutic effect of T-DM1 because it plays a key role in regulating T-DM1 internalization.

Overexpression of suppressor of IKBKE 1 is associated with vincristine resistance in colon cancer cells.

In a previous study, the suppressor of IKBKE 1 expression level was confirmed to be higher in vincristine (VCR)-resistant HCT-8 (HCT-8/V) colon cancer cells than in non-VCR-resistant HCT-8 cells. In the current study, IKBKE 1 expression in VCR-resistant colon cancer cells was investigated further. HCT-8 and HCT-8/V human colon cancer cells were used, and polymerase chain reaction (PCR) primers were designed to amplify the IKBKE 1 gene. Fluorescence reverse transcription-quantitative PCR (RT-qPCR) was performed to detect differences in IKBKE 1 expression between sensitive and drug-resistant colon cancer cell lines. Western blotting was performed to further observe IKBKE 1 expression. Based on the RT-qPCR and western blot results, IKBKE 1 expression was observed to be markedly higher in the HCT-8/V cells, and this difference was significant (P<0.05). Thus, IKBKE 1 expression was identified to be associated with the resistance of colon cancer cells to VCR.