The author acknowledges Dr. Christian Derntl from the Department for Biotechnology and Microbiology at the TU Wien and the Department of Medicine III, Division of Nephrology and Dialysis at the Medical University of Vienna, especially Dr. Markus Wahrmann for technical and scientific support. Protein expression in mammalian cells was supported by the Department of Biotechnology at the University of Natural Resources and Life Sciences (BOKU) Vienna. The author wants to express her deep acknowledgement to Dr. Nico Dankbar from XanTec bioanalytics in Duesseldorf, Germany, for helpful scientific discussions on performing the SPR binding assays.

For the present study, a CVN-small ubiquitin-like modifier (SUMO) fusion protein has been used in enzyme-linked immunosorbent assays instead of CV-N and is suitable for cell-based assays. Recombinant full-length influenza A virus HA H3 protein is obtained commercially (see Table of Materials) or expressed in mammalian HEK293 cell lines and baculovirus-infected insect cells according to standard protocols12. Wuhan-1 spike protein is expressed in mammalian HEK293 cells. The synthesi...

CV-N&#39;s binding affinity is correlated with the number of functional binding sites [2H on domains B, and 2L on domain(s) A when engineered as domain-swapped dimer]. A variant with an altered binding affinity (CVN2L0-V2, a homodimeric stable fold of CV-N comprising a disulfide bridge knock-out) is expressed in E. coli, purified, and positively tested for binding to HA-protein (H3N2) using SPR10, and shows a conformational change upon binding HA with either H or L carbohydrate-binding si...

Tetherin-associated antiviral activity is induced by interferon-&#945;, and it comprises protein-based tethers, that leads to the retention of fully formed virions on infected cell surfaces1. The necessity for tetherin glycosylation in the inhibition of virus release remains uncertain, implying the importance of glycosylation patterns on recombinantly expressed glycans for in vitro studies1,2, which depends on the conformation of (in the case of influenza virus) surface-expressed influenza hemagglutinin HA3,4. It has been noted that modification of oligosaccharide tethered to N-linked glycosylation is enough for tetherin-mediated restriction of HIV type-1 release2, while dimerization plays an essential role in preventing virus release, thereby involving the transmembrane domain or glycosyl-phosphatidyl-inositol (GPI)-anchor for tethering the budding virions5. Unique features are described for human and murine tetherin to block multiple enveloped viruses, retroviruses, and filoviruses. BST-2/tetherin is an interferon-inducible antiviral protein of the innate immunity1,6, acting with broad-spectrum antiviral activity and is antagonized by envelope glycoproteins5 to either translocate tetherin or disrupt the structure of tetherin6. For example, surface-expressed envelope glycoprotein HA and neuraminidase on influenza A virus are well known for tetherin antagonism in a strain-specific manner7, facilitating the recognition of host receptor binding sites8. Glycan-targeting antibodies are studied in the stoichiometry of their interactions with the rapidly customizing glycan shields on HA, resulting in binding affinity to influenza A H3N2 and H1N1 subtypes4.
To elucidate the binding mechanisms between antiviral agents and virus envelope spikes, i.e., carbohydrate ligands, and complementary immunological and spectroscopic methods, mono-, di- and tri-mannose moieties are chemically synthesized. The mannosylated peptides are created via azido glycosylation of glycosyl {beta}-peracetates to 1,2-trans glycosyl azides transformation9, mimicking the typically found N-acetyl glucosamine and high-mannose oligosaccharides on the surface of life-threatening viruses. Triazole bioisosteres are utilized to mimic linkages that form the mannosylated residue of HA peptide10 and facilitate site-specific interactions with antiviral CV-N derivatives around the second N-linked glycosylation spot on the HA head domain (HA top with 4 N-linked glycans N54, N97, N181, N301)8,11,12. Interactions between glutamic acid (Glu) and arginine (Arg) and the resulting helix dipole manifestated good stability of both model peptides and proteins but are visualized using SPR. If compared with recognizing a single chemically synthesized glycosylation site on HA10 by directly inhibiting receptor binding on the glycan moieties, a higher affinity of a four-site mutated Fc structure to its receptor is shown to elicit effector functions in vivo, revealing the unrelated composition of N-linked glycans attached to Fc mutant to be mechanistically determined13.
CV-N displays antiviral activity against HIV14,15, influenza16, and Ebola virus, which is mediated by nanomolar binding to high-mannose oligosaccharide modifications on envelope spike proteins12,17,18,19. Influenza HA binding to one high-affinity carbohydrate-binding site (H) in CV-N or two Hs in covalently linked dimeric CVN2 is determined to have equilibrium dissociation constants (KD) = 5.7 nM (Figure 1A) and KD = 2.7 nM, respectively. Both CV-N and CVN2 harbor another one or two low-affinity carbohydrate-binding sites (L)s12,17,20,21. Ebola GP1,2 binds to 2H of CVN2 with affinities in the lower nanomolar range (KD = 26 nM). CV-N WT binding to Ebola GP1,2 and HA exhibits affinities from KD = 34 nM to KD = 5.7 nM (A/New York/55/04)12. Lectins, such as CV-N, which specifically target high-mannose glycans on the viral envelopes, further inhibit replication of hepatitis C virus, SARS-CoV, herpesvirus, Marburg virus, and measles virus22.
The small CV-N molecule has been studied thoroughly for more than 20 years as it functionalizes to bind a wide range of viruses to inhibit viral entry16,18. Structural analyses and binding affinity assays indicate cross-linking of two Ls in a domain-swapped CVN2 dimer by bivalent binding in the micromolar range to enhance avidity to viral envelope glycoproteins10,19. Selective binding of Man&#945;1-2Man&#945; on Man(8) D1D3 arms and Man(9) comprises two binding sites of differing affinities located on opposite protein protomers20, thereby reaching nanomolar binding affinities (Figure 1B). Thus, CVN2 is considered a pseudo-antibody concerning its application to bind epitopes on HIV gp120, similar to virus-neutralizing antibodies17. Herein, the author is interested in investigating the potential binding of CVN2 to the SARS-CoV-2 spike via its receptor-binding domain (RBD). Binding curves of immobilized human angiotensin-converting enzyme (ACE)-2 with the SARS-CoV-2 RBD result in KD = 4.7 nM for this biologically relevant binding interaction23.
By contrast, selected immunoglobulin classes recognize specific and consistent structural protein patterns, which impart a substrate for affinity maturation in the membrane-anchored HA regions24. CV-N shows highly potent activity in almost all influenza A and B viruses16, and it is a broadly neutralizing antiviral agent. Our knowledge is incomplete on the location of targeted epitopes on the stem of HA1 and HA2 that possibly involve epitopic structures for glycan-targeting by highly neutralizing antibodies and as compared with lectin binding25.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/63541/63541fig1v5.jpg" /> 
Figure 1: Schematic representation of the SPR binding assay for CV-N to virus envelope spikes. (A) SPR Assay for CV-N binding to ligand: HA full-length protein (90 kDa). Kinetic data set (5120, 2560, 1280, 640, 320, 160, 80, 40, 20, 10, 5, 2.5, 0 nM) showing real-time double-referenced binding to influenza HA A/New-York/55/04 (H3N2). (B) CVN2L0 variant V2 binding to immobilized ligand DM within a concentration range of 500 nM to 16 &#181;M. Sequence: L residues are highlighted in yellow. H residues are highlighted in gray. E58 and R73 are a replacement for cysteines in the wildtype protein and make V2 a stable protein fold with three instead of four disulfide bonds <a href="https://www.jove.com/files/ftp_upload/63541/63541fig1v5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Whereas the glycan shield on the membrane-distal HA top part induces high-affinity binding to CV-N12, CVN2 binding to HA adjacent to a disulfide bridge of the HA top part has further been observed at its low-affinity sites10,12. Various polar interactions and interaction sites are identified in carbohydrate-binding by CV-N. These interactions are verified by generating knock-out variants in the binding site to correlate binding affinities to in silico predicted glycosylation12. Thus, the project aims to compare previously tested chemically mannosylated HA peptides in binding affinity and specificity with short peptide sequences from SARS-related 2019-nCoV spikes and SARS-CoV-2, naturally occurring modified by a small number of different N-linked glycosylation sites and O-linked glycosylation. Using cryo-electron microscopy and binding assays, Pinto and coworkers report a monoclonal antibody, S309, that potentially recognizes an epitope on SARS-CoV-2 spike protein containing a conserved glycan within the Sarbecovirus subgenus, without competing with receptor attachment26. The protocol of this study describes how designing, expressing, and characterizing CV-N variants are important to study how CV-N and CVN2 bind to glycosylated proteins and synthetic mannosylated peptides using the SPR technology10,12.
Tandem-linked dimer CVN2L027 and binding-site variants (V2-V5) are recombinantly expressed and variants are with disulfide bond replacements (C58E and C73R) (Figure 2A). Also, a mutant with a single-point mutation E41A is prepared because this position has been seen as an intermolecular cross-contacting residue. This mutant is another interesting molecule for SPR binding measurements between the lectin and high-mannose oligosaccharides deciphering binding domains and allows comparison with the dimeric form. The domain-swapped crystal structure of CVN2 shows a flexible linker, that extends between 49 and 54 residues. The two domains can continue moving around the hinge as rigid bodies, developing either a monomer through intramolecular domain interactions (domain A -residues 1-39;90-101- with domain B -residues 40-89) or a dimer by intermolecular domain swapping [domain A (of the first monomer) with domain B (of the second), and domain B (of the first monomer) with domain A (of the second copy)]. There are no close interactions between the two protomers&#39; A and B domains, except for Glu4128. The gene for CV-N can be developed using a repetitive PCR method with 40-mer synthesized oligos29 and is then subcloned into the NdeI and BamHI sites of pET11a for transformation (electroporation) into electrocompetent cells as described by Keeffe, J.R.27. The protein, which is used for achieving the respective crystal structure (PDB ID 3S3Y), includes an N-terminal 6-histidine purification tag followed by a Factor Xa protease cleavage site. Site-directed mutagenesis is utilized to make point mutations, switch codons, and insert or delete single or multiple bases or codons for amino acid exchange. These transformations provide invaluable insight into protein function and structure. Recombinantly expressed and purified CV-N, CVN2, and CVN3 have been biophysically well studied20,21,27, are cheap to produce, and therefore used to characterize binding assays to glycans immobilized onto SPR sensor chips. Conventional enzyme-linked immunosorbent assay (ELISA) provides less reproducibility concerning the immobilization technique of glycan ligands and transforms real-time binding of various binding-site variants, which is shown for SPR, into endpoint assays.
Binding-affinity variant CVN2L0-V2 (an intact fold of homodimeric CV-N with a disulfide bridge substitution10) is expressed with a His-tag in Escherichia coli (E. coli), purified over Ni-NTA column applying affinity chromatography and tested for binding to HA (H3N2), monomannosylated HA-peptide and dimannosylated HA-peptide using SPR. Chemically mannosylated peptides, or HA and S protein, all are ligands and amine coupled to the hydrophilic chip surface via reactive esters or biotin-streptavidin protein engineering. The same procedure of sequential runs is applied to those ligands, injecting various dilutions of CV-N and variants of CV-N (and CVN2) to obtain kinetic information for the molecular interaction analyses as described below30. RBD-immobilized SPR sensor chip is used for binding studies on CV-N to S peptides, and affinities are compared to SARS-CoV-2 binding with the human ACE2.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>&#196;kta primeplus</td>
			<td>Cytiva</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Amicon tubes</td>
			<td>Merck</td>
			<td>C7715</td>
			<td></td>
		</tr>
		<tr>
			<td>Ampillicin</td>
			<td>Sigma-Aldrich</td>
			<td>A5354</td>
			<td></td>
		</tr>
		<tr>
			<td>Beckmann Coulter Cooler Allegra X-30R centrifuge</td>
			<td>Beckman Coulter</td>
			<td>B06320</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell spreader</td>
			<td>Sigma-Aldrich</td>
			<td>HS86655</td>
			<td>silver stainless steel, bar L 33&#160;mm</td>
		</tr>
		<tr>
			<td>Custom DNA Oligos</td>
			<td>Sigma-Aldrich</td>
			<td>OLIGO</td>
			<td></td>
		</tr>
		<tr>
			<td>Custom Gensynthesis</td>
			<td>GenScript</td>
			<td>#1390661&#160;</td>
			<td>cloning vector: pET27b(+)&#160;</td>
		</tr>
		<tr>
			<td>Cytiva&#160;HBS-EP+ Buffer 10, 4x50mL</td>
			<td>Thermo Scientific</td>
			<td>50-105-5354</td>
			<td></td>
		</tr>
		<tr>
			<td>Dionex UlitMate 3000</td>
			<td>Thermo Scientific</td>
			<td>IQLAAAGABHFAPBMBFB</td>
			<td></td>
		</tr>
		<tr>
			<td>Dpn I restriction enzyme (10 U/&#956;L)&#160;</td>
			<td>Fisher Scientific</td>
			<td>ER1701</td>
			<td></td>
		</tr>
		<tr>
			<td>DTT</td>
			<td>Merck</td>
			<td>DTT-RO</td>
			<td></td>
		</tr>
		<tr>
			<td>EDC</td>
			<td>Merck</td>
			<td>39391</td>
			<td></td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>Merck</td>
			<td>E9884</td>
			<td></td>
		</tr>
		<tr>
			<td>Eppendorf Safe-Lock Tubes</td>
			<td>Eppendorf</td>
			<td>30120086</td>
			<td></td>
		</tr>
		<tr>
			<td>Eppendorf Safe-Lock Tubes</td>
			<td>Eppendorf</td>
			<td>30120094</td>
			<td></td>
		</tr>
		<tr>
			<td>Eppendorf&#160;Minispin&#160;and MiniSpin Plus personal microcentrifuge</td>
			<td>Sigma-Aldrich</td>
			<td>Z606235</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>Merck</td>
			<td>51976</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanolamine HCl</td>
			<td>Merck</td>
			<td>E6133</td>
			<td></td>
		</tr>
		<tr>
			<td>Falcon 50mL Conical Centrifuge Tubes</td>
			<td>Fisher Scientific</td>
			<td>14-432-22</td>
			<td></td>
		</tr>
		<tr>
			<td>Falcon 14 mL Round Bottom Polystyrene Test Tube, with Snap Cap, Sterile, 25/Pack</td>
			<td>Corning</td>
			<td>352057</td>
			<td></td>
		</tr>
		<tr>
			<td>Glucose</td>
			<td>Merck</td>
			<td>G8270</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycine HCl</td>
			<td>Merck</td>
			<td>55097</td>
			<td></td>
		</tr>
		<tr>
			<td>HA H3 protein</td>
			<td>Abcam</td>
			<td>ab69751</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Merck</td>
			<td>H3375</td>
			<td></td>
		</tr>
		<tr>
			<td>His-select Ni2+</td>
			<td>Merck</td>
			<td>H0537</td>
			<td></td>
		</tr>
		<tr>
			<td>Imidazole</td>
			<td>Merck</td>
			<td>I2399</td>
			<td></td>
		</tr>
		<tr>
			<td>IPTG</td>
			<td>Merck</td>
			<td>I6758</td>
			<td></td>
		</tr>
		<tr>
			<td>Kanamycin A</td>
			<td>Sigma-Aldrich</td>
			<td>K1377</td>
			<td></td>
		</tr>
		<tr>
			<td>Kromasil 300-5-C4</td>
			<td>Nouryon</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>LB agar</td>
			<td>Merck</td>
			<td>52062</td>
			<td></td>
		</tr>
		<tr>
			<td>LB agar</td>
			<td>Merck</td>
			<td>19344</td>
			<td></td>
		</tr>
		<tr>
			<td>LB Lennox</td>
			<td>Merck</td>
			<td>L3022</td>
			<td></td>
		</tr>
		<tr>
			<td>Lysozyme</td>
			<td>Merck</td>
			<td>10837059001</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnesium chloride</td>
			<td>Merck</td>
			<td>M8266</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnesium sulfate</td>
			<td>Merck</td>
			<td>M7506</td>
			<td></td>
		</tr>
		<tr>
			<td>NaH2P04</td>
			<td>Merck</td>
			<td>S0751</td>
			<td></td>
		</tr>
		<tr>
			<td>NanoDrop UV-Vis2000c spectrophotometer</td>
			<td>Thermo Scientific</td>
			<td>ND2000CLAPTOP</td>
			<td></td>
		</tr>
		<tr>
			<td>NaOH</td>
			<td>Merck</td>
			<td>S5881</td>
			<td></td>
		</tr>
		<tr>
			<td>NHS</td>
			<td>Merck</td>
			<td>130672</td>
			<td></td>
		</tr>
		<tr>
			<td>NZ amine (casein hydrolysate)</td>
			<td>Merck</td>
			<td>C0626</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Merck</td>
			<td>806552</td>
			<td></td>
		</tr>
		<tr>
			<td>PD MidiTrap G-10</td>
			<td>Sigma-Aldrich</td>
			<td>GE28-9180-11</td>
			<td></td>
		</tr>
		<tr>
			<td>Peptone</td>
			<td>Merck</td>
			<td>70171</td>
			<td></td>
		</tr>
		<tr>
			<td>pET11a</td>
			<td>Merck Millipore (Novagen)</td>
			<td>69436&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>PMSF</td>
			<td>Merck</td>
			<td>PMSF-RO</td>
			<td></td>
		</tr>
		<tr>
			<td>QIAprep Spin Miniprep Kit (1000)</td>
			<td>Qiagen</td>
			<td>27106X4</td>
			<td></td>
		</tr>
		<tr>
			<td>Reichert Software Package Autolink1-1-9</td>
			<td>Reichert</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Reichert SPR SR7500DC Dual Channel System</td>
			<td>Reichert</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Scrubber2-2012-09-04 for data analysis</td>
			<td>Reichert</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>SDS</td>
			<td>Merck</td>
			<td>11667289001</td>
			<td></td>
		</tr>
		<tr>
			<td>Site-directed mutagenesis kit incl pUC18 control plasmid</td>
			<td>Stratagene</td>
			<td>#200518</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodim chloride</td>
			<td>Merck</td>
			<td>S9888</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium acetate.Trihydrate</td>
			<td>Merck</td>
			<td>236500</td>
			<td></td>
		</tr>
		<tr>
			<td>SPR sensor chip C19RBDHC30M</td>
			<td>XanTec bioanalytics</td>
			<td>SCR C19RBDHC30M</td>
			<td></td>
		</tr>
		<tr>
			<td>SPR sensor chip CMD500D</td>
			<td>XanTec bioanalytics</td>
			<td>SCR CMD500D</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterilin Standard 90mm Petri Dishes</td>
			<td>Thermo Scientific</td>
			<td>101R20</td>
			<td></td>
		</tr>
		<tr>
			<td>TBS</td>
			<td>Merck</td>
			<td>T5912</td>
			<td>10x, solution</td>
		</tr>
		<tr>
			<td>Triton-X100</td>
			<td>Merck</td>
			<td>T8787</td>
			<td></td>
		</tr>
		<tr>
			<td>Tryptone</td>
			<td>Merck</td>
			<td>93657</td>
			<td></td>
		</tr>
		<tr>
			<td>Tween20</td>
			<td>Merck</td>
			<td>P1379</td>
			<td></td>
		</tr>
		<tr>
			<td>Vortex-Genie 2 Mixer</td>
			<td>Merck</td>
			<td>Z258423</td>
			<td></td>
		</tr>
		<tr>
			<td>X-gal</td>
			<td>Merck</td>
			<td>XGAL-RO</td>
			<td></td>
		</tr>
		<tr>
			<td>XL1-Blue Supercompetent Cells</td>
			<td>Stratagene</td>
			<td>#200236</td>
			<td></td>
		</tr>
		<tr>
			<td>Yeast extract</td>
			<td>Merck</td>
			<td>Y1625</td>
			<td></td>
		</tr>
	</tbody>
</table>

engineering antiviral agents via surface plasmon resonance

Surface plasmon resonance (SPR) is used to measure hemagglutinin (HA) binding to domain-swapped Cyanovirin-N (CV-N) dimer and to monitor interactions between mannosylated peptides and CV-N's high-affinity binding site. Virus envelope spikes gp120, HA, and Ebola glycoprotein (GP) 1,2 have been reported to bind both high- and low-affinity binding sites on dimeric CVN2. Dimannosylated HA peptide is also bound at the two low-affinity binding sites to an engineered molecule of CVN2, which is bearing a high-affinity site for the respective ligand and mutated to replace a stabilizing disulfide bond in the carbohydrate-binding pocket, thus confirming multivalent binding. HA binding is shown to one high-affinity binding site of pseudo-antibody CVN2 at a dissociation constant (KD) of 275 nM that further neutralizes human immunodeficiency virus type 1 (HIV-1) through oligomerization. Correlating the number of disulfide bridges in domain-swapped CVN2, which are decreased from 4 to 2 by substituting cystines into polar residue pairs of glutamic acid and arginine, results in reduced binding affinity to HA. Among the strongest interactions, Ebola GP1,2 is bound by CVN2 with two high-affinity binding sites in the lower nanomolar range using the envelope glycan without a transmembrane domain. In the present study, binding of the multispecific monomeric CV-N to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike (S) glycoprotein is measured at KD = 18.6 &#181;M as compared with nanomolar KD to those other virus spikes, and via its receptor-binding domain in the mid-&#181;-molar range.

A dimeric domain-swapped CVN2L0 molecule is tested for binding to the HA top region in three separate SPR experiments and binding affinity is presented in KD values. Domain B is assumed to comprise H-binding sites, which are impacted by replacing a disulfide bond into ionic residues, and domain A forms L10,18. Single injections of CVN2L0 and variants V2 (three disulfide bridges) and V5 (two disulfide bridges) are first tested for binding to the HA-coup...

Watch this Scientific Journal Video about Engineering Antiviral Agents via Surface Plasmon Resonance at JoVE.com

Engineering Antiviral Agents via Surface Plasmon Resonance

The present protocol describes new tools for SPR binding assays to examine CV-N binding to HA, S glycoprotein, related hybrid-type glycans, and high-mannose oligosaccharides. SPR is used to determine the KD for binding either dimeric or monomeric CV-N to these glycans.

Engineering Antiviral Agents via Surface Plasmon Resonance

Surface plasmon resonance (SPR) is used to measure hemagglutinin (HA) binding to domain-swapped Cyanovirin-N (CV-N) dimer and to monitor interactions ...

Surface plasmon resonance spectroscopy has been established for the determination of protein ligand in a medium throughput way. Different affinities for binding site variants of the same molecule are presented, namely for the of iron-binding high manners oligosaccharides on viruses. SPR is a label-free technique to study real-time macromolecular binding of surface immobilized receptors.Multisite interactions are recognized in the kinetic analysis by competitive binding independently of the size of the Specific areas in research that use SPR are drug development to search for inhibitors or antibody characterization. Kinetic constants and affinities are directly calculated from the sensor response. To begin, transfer 100 microliters of the solution on LB-agar plates and spread it gently using a sterile cell spreader.Start a series of sample reactions using multiple concentrations of double-stranded DNA template ranging from five to 50 nanograms while keeping the primer concentration constant. Then add the DpnI restriction enzyme below the mineral oil overlay and incubate immediately at 37 degrees Celsius for one hour for digesting the parental supercoiled double-stranded DNA. To thaw the XL1-Blue Supercompetent Cells, aliquot the cells to a pre-chilled, small, round bottom tube.Next, transfer one microliter of the DpnI treated single-stranded DNA from each control and sample reaction to separate aliquots of the Supercompetent Cells which synthesize the complementary strand. After swirling the transformation reactions carefully to mix, incubate the reactions on ice for 30 minutes. Apply heat pulse to the transformation reactions at 42 degrees Celsius for 45 seconds and then place the reactions on ice for two minutes.Then add 0.5 milliliters of NZY+Broth and incubate the transformation reactions at 37 degrees Celsius with shaking at 225 to 250 RPM for one hour. Afterward, plate the correct volume of each transformation reaction on LB Ampicillin Agar plates as demonstrated previously. For a seed culture, inoculate a small amount of ampicillin containing LB media with a single colony from the transformed plate.Using the overnight culture, inoculate the expression culture with additives including 10 millimolar of magnesium chloride, 10 millimolar of magnesium sulfate, and 20 millimolar of glucose, diluting the seed culture to one by 100. Next, grow cells with vigorous shaking at 37 degrees Celsius to an absorbent 600 nanometer between 0.4 to 0.6 before cooling the cells to 20 degrees Celsius and induce with one millimolar IPTG and grow overnight. Then harvest the cells by centrifuging at 4, 000 g for 15 minutes at four degrees Celsius and discard the supernatant.After resuspending the cell pellet in phosphate buffered saline buffer, recent refuge at 4, 000 g for 15 minutes at four degrees Celsius. Then discard the supernatant with a pipette. Next, resuspend the remaining pellet in 10 milliliters of lysis buffer and incubate the suspension for one hour at 37 degrees Celsius.Then separate soluble and insoluble fractions by centrifugation at 4, 000 g for 15 minutes at four degrees Celsius. Additionally, use HIS-Select Ni2+Affinity Gel in 14 milliliters tubes to bind and resuspend his tagged recombinantly expressed cyanovirin-N in buffer solutions with 20 millimolar imidazole and 250 millimolar imidazole, respectively. Incubate in batch for at least 30 minutes between these steps.Adding the elution buffer containing 250 millimolar imidazole to elute protein. Transfer the protein solutions to centrifugation tubes with a 10 kilo Dalton cutoff filter and concentrate them by centrifuging for 10 minutes at 4, 500 g and four degrees Celsius. Add SPR running buffer to a dilution factor of one to 10 and centrifuge four times to the initial volume for 10 minutes at 4, 500 g and four degrees Celsius.Afterward, determine the protein concentration at 280 nanometer using a NanoDrop UV-Visible Spectrophotometer based on the calculated extinction co-efficient for the main protein CVN2L0 showing a size of 23, 474 Dalton. For the dual channel SPR system, use HBSCP plus as running buffer, 10 millimolar glycine hydrochloric acid of pH 1.5 to 1.6 as the regeneration buffer and turn on the instrument degasser, autosampler and pump. Then wash the entire system with double distilled water for one hour.Next, drop immersion oil onto the detector and mount a glass sensor chip coated with a thin gold film. And on the upper side, functionalized with carboxymethyl dextran hydrogel directly onto the detector below the three-port flow cell. Then fix the setting by pulling down the handling.To immobilize the proteinaceous ligands to sensor chips, open a run table by clicking on Form in the menu bar and run table editor in the integrated SPP auto link software. Then choose and click on Basic Immobilization from the list of available run tables and follow the steps of the experimental procedure on the computer screen. Next, click on Sample Set Editor in the form section to fill out the reagents list for two racks placed in the autosampler for further analysis.Click on Autosampler Direct Control as a tool in the menu bar to bring the racks forward or back home and choose four degrees Celsius as the operating temperature. Start the pump to infuse double distilled water by clicking on tools and pump direct control. And record data by clicking on SPR Instrument Direct Control.And each time, start in the newly appearing window. After adding the coupling reagents in 300 microliter vials, put them into the autosampler racks and start the run table by clicking on Run. For the simple protein-protein interaction, after refilling the pump at 25, 000 microliters per minute, perform baseline adjustment for 30 seconds.Allow for the subsequent baseline adjustment with double distilled water for 1.5 minutes before quenching the activated chip surface with one molar ethanolamine hydrochloride, pH 8.5. Then switch the tubes from the liquid sampler to the degasser from double distilled water into the bottle with HBS-EP+Click on Form, scroll down, and change to Post-Processing by clicking this operation mode. Then click on Add to select binding curves generated over time in the data platform for each flow cell and export the overlay as a scrubber file.Next, click on File to open the file saving options and obtain response curves by aligning left and right curves and subtracting signals of the second reference channel from those of the ligand channel. Single injections of CVN2L0 and variance V2 and V5 were first tested for binding to the haemagglutinin coupled sensor chip to estimate binding capacities using the autosampler and running table editor. Repeated injections were automated at various concentrations in the nanomolar and micromolar range in the system over time demonstrating that no saturation on the chip surface nor equilibrium binding was achieved.Concentration versus response was plotted for CVN wild-type binding to RBD, assuming specific targeting of possibly conserved carbohydrates on the RBD S1 subunit with weaker affinity having a dissociation rate constant of 260 micromoles. The same affinity plot for CVN2L0-V4 binding to DM becomes no longer analyzable due to the replacement of disulphide bonds that interfere with high-affinity binding to small glycosylated peptides and yields a higher response than response maximum value. CVN wild-type binding to RBD on SARS-CoV-2 shows binding to S protein and RBD with a dissociation rage constant of 18.6 micromoles and 260 micromoles, respectively.Although SPR response for analyte concentrations results in high response units and typical SPR sensorgrams for CVN wild-type and E41A binding. However, the mutant is unstable when diluted. SPR biosensing is addressing high-affinity and low-affinity binding of purified proteins and its variants to ligands.In our case, glycoproteins exploiting optic readout and the dual channel flow system on sensor chip surface. Enzyme being immunosorbent assay is an alternate immunological methods recognizing protein epitopes but also providing data on the concentration of peptides and small molecules out of complex matrices. Testing recombinantly expressed proteins by SPR binding assays incorporated biosensing of confirmational changes which is useful for the verification and prediction of protein stability by computational protein side.

engineering-antiviral-agents-via-surface-plasmon-resonance

Research

JoVE Journal

Bioengineering

2.1K visualizações.  University of California, Los Angeles. A espectroscopia de ressonância plasmônica de superfície foi estabelecida para a determinação do ligante proteico de forma de rendimento médio. Diferentes afinidades para variantes de sítios de ligação da mesma molécula são apresentadas, nomeadamente para a ligação de ferro a oligossacarídeos de alta qualidade em vírus. A SPR é uma técnica livre de rótulos para estudar a ligação macromolecular em tempo real de receptores imobilizados de superfície.As interações m...