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.

Engineered scPDL1-DM1 drug conjugate with improved in vitro analysis to target PD-L1 positive cancer cells and intracellular trafficking studies in cancer therapy.

Antibody-drug conjugates (ADC), precisely deliver a cytotoxic agent to antigen-expressing tumor cells by using specific binding strategies of antibodies. The ADC has shown the ability of potent bio-therapeutics development but indefinite stoichiometric linkage and full-length antibody penetration compromised the field of its advancement. Single chain variable fragments convention instead of the full-length antibody may overcome the challenge of rapid penetration and internalization. Programmed cell death ligand-1 interaction with PD-1 has recently revolutionized the field of immunotherapy. We systematically designed scPDL1-DM1 drug conjugate by linking scFv-PD-L1 proteins (scFv) with maytansinoids (DM1) cytotoxic agent through succinimidyl trans-4-maleimidylmethyl cyclohexane-1- carboxylate (SMCC) linker. Binding affinity was confirmed by immunocytochemistry, spectrophotometry and gel electrophoresis analysis. The scPDL1-DM1 showed specific binding with PD-L1 positive tumor cells and retained in vitro anti-cell proliferation activity. The intracellular trafficking of the drug was evaluated in A549 cancer cell lines, and maximum trafficking was observed after two hours of incubation. The generated drug can be utilized as a potent tool for site-specific conjugation, predicting specificity in vitro activities with extended range against PD-L1 positive cancer cells and can be utilized for further in vivo testing and clinical therapeutics development.

Efficient development and expression of scFv recombinant proteins against PD-L1 surface domain and potency in cancer therapy.

PD-L1 is a 40 kDa trans-membrane protein of B7 family and an important T cell regulator. Binding of PD-L1 and PD-1 inhibits proliferation and activation of T cell results cell exhaustion. This phenomenon can be reversed by blocking PD-L1/PD-1 interactions with single chain variables fragment (scFv) fusion proteins and by direct inhibition of tumor cells with drug conjugates. The human phage-displayed scFv library was utilized to generate scFv against the PD-L1 antigen by affinity bio-panning. The positive clones were selected by continuous transfection of bacterial cells and sequence analysis. The binding affinity and specificity of the scFv and antibody fragments were determined by using surface plasmon resonance biosensor, western blot analysis, and immunofluorescence assay. After three rounds of panning selection, about 30% of clones have a binding affinity with targeted PD-L1 antigen. Eight positive clones with accurate sequences were isolated and analyzed for binding affinity with PD-L1 antigen. Three of those with accurate sequences and binding affinity were selected for the recombinant formation and soluble expression by Escherichia coli host machinery. The highly positive recombinant clones with the exact orientation of FR and CDR domains were developed and can be used as a drug carrier tools in ADC formation or direct inhibition of immune checkpoint in cancer immunotherapy. The conjugate achieved its initial potency and need efficient improvement to enhance direct tumor suppression and bio-therapeutics strategies enrichment.