Name: a competent hepatocyte model examining hepatitis b virus entry through sodium taurocholate cotransporting polypeptide as a therapeutic target
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This research project is supported by Mahidol University and the Thailand Science Research and Innovation (TSRI) separately awarded to A. Wongkajornsilp and K. Sa-ngiamsuntorn. This work was financially supported by the Office of National Higher Education Science Research and Innovation Policy Council through the Program Management Unit for Competitiveness (grant number C10F630093). A. Wongkajornsilp is a recipient of a Chalermprakiat grant of the Faculty of Medicine Siriraj Hospital, Mahidol University. The authors would like to thank Miss Sawinee Seemakhan (Excellent Center for Drug Discovery, Faculty of Science, Mahidol University) for her assistance with the ITC technique.

NOTE: The following procedures must be performed in a Class II biological hazard flow hood or a laminar-flow hood. The handling of HBV was ethically approved by the IRB (MURA2020/1545). See the Table of Materials for details about all solutions, reagents, equipment, and cell lines used in this protocol.
1. Preparing host cells (mature hepatocytes)
<ol>
	<li>Culture hepatocytes (3.75 &#215; 105 cells HepaRG or imHC) and maintain in a 75 cm2</s...

<ol>
	<li>Levrero, M., Zucman-Rossi, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+of+HBV-induced+hepatocellular+carcinoma.">Mechanisms of HBV-induced hepatocellular carcinoma.</a> Journal of Hepatology. 64 (1), 84-101 (2016).</li><li>Kim, K. -. H., Kim, N. D., Seong, B. -. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Discovery+and+development+of+anti-HBV+agents+and+their+resistance.">Discovery and development of anti-HBV agents and their resistance.</a> Molecules. 15 (9), 5878-5908 (2010).</li><li>Shaw, T., Bowden, S., Locarnini, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemotherapy+for+hepatitis+B:+New+treatment+options+necessitate+reappraisal+of+traditional+endpoints.">Chemotherapy for hepatitis B: New treatment options necessitate reappraisal of traditional endpoints.</a> Gastroenterology. 123 (6), 2135-2140 (2002).</li><li>Volz, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+entry+inhibitor+Myrcludex-B+efficiently+blocks+intrahepatic+virus+spreading+in+humanized+mice+previously+infected+with+hepatitis+B+virus.">The entry inhibitor Myrcludex-B efficiently blocks intrahepatic virus spreading in humanized mice previously infected with hepatitis B virus.</a> Journal of Hepatology. 58 (5), 861-867 (2013).</li><li>Mak, L. -. Y., Seto, W. -. K., Yuen, M. -. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+antivirals+in+clinical+development+for+chronic+hepatitis+B+infection.">Novel antivirals in clinical development for chronic hepatitis B infection.</a> Viruses. 13 (6), 1169 (2021).</li><li>Zuccaro, V., Asperges, E., Colaneri, M., Marvulli, L. N., Bruno, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=HBV+and+HDV:+New+Treatments+on+the+Horizon.">HBV and HDV: New Treatments on the Horizon.</a> Journal of Clinical Medicine. 10 (18), 4054 (2021).</li><li>Iwamoto, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+and+identification+of+hepatitis+B+virus+entry+inhibitors+using+HepG2+cells+overexpressing+a+membrane+transporter+NTCP.">Evaluation and identification of hepatitis B virus entry inhibitors using HepG2 cells overexpressing a membrane transporter NTCP.</a> Biochemical and Biophysical Research Communications. 443 (3), 808-813 (2014).</li><li>Tong, S., Li, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+NTCP+as+an+HBV+receptor:+the+beginning+of+the+end+or+the+end+of+the+beginning.">Identification of NTCP as an HBV receptor: the beginning of the end or the end of the beginning.</a> Gastroenterology. 146 (4), 902-905 (2014).</li><li>Xuan, J., Chen, S., Ning, B., Tolleson, W. H., Guo, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+HepG2-derived+cells+expressing+cytochrome+P450s+for+assessing+metabolism-associated+drug-induced+liver+toxicity.">Development of HepG2-derived cells expressing cytochrome P450s for assessing metabolism-associated drug-induced liver toxicity.</a> Chemico-Biological Interactions. 255, 63-73 (2016).</li><li>Thongsri, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Curcumin+inhibited+hepatitis+B+viral+entry+through+NTCP+binding.">Curcumin inhibited hepatitis B viral entry through NTCP binding.</a> Scientific Reports. 11 (1), 19125 (2021).</li><li>Sa-Ngiamsuntorn, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+immortalized+hepatocyte-like+cell+line+(imHC)+accommodated+complete+viral+lifecycle,+viral+persistence+form,+cccDNA+and+eventual+spreading+of+a+clinically-isolated+HBV.">An immortalized hepatocyte-like cell line (imHC) accommodated complete viral lifecycle, viral persistence form, cccDNA and eventual spreading of a clinically-isolated HBV.</a> Viruses. 11 (10), 952 (2019).</li><li>Watashi, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cyclosporin+A+and+its+analogs+inhibit+hepatitis+B+virus+entry+into+cultured+hepatocytes+through+targeting+a+membrane+transporter,+sodium+taurocholate+cotransporting+polypeptide+(NTCP).">Cyclosporin A and its analogs inhibit hepatitis B virus entry into cultured hepatocytes through targeting a membrane transporter, sodium taurocholate cotransporting polypeptide (NTCP).</a> Hepatology. 59 (5), 1726-1737 (2014).</li><li>Kaneko, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+tricyclic+polyketide,+Vanitaracin+A,+specifically+inhibits+the+entry+of+hepatitis+B+and+D+viruses+by+targeting+sodium+taurocholate+cotransporting+polypeptide.">A novel tricyclic polyketide, Vanitaracin A, specifically inhibits the entry of hepatitis B and D viruses by targeting sodium taurocholate cotransporting polypeptide.</a> Journal of Virology. 89 (23), 11945-11953 (2015).</li><li>Manta, B., Obal, G., Ricciardi, A., Pritsch, O., Denicola, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tools+to+evaluate+the+conformation+of+protein+products.">Tools to evaluate the conformation of protein products.</a> Biotechnology Journal. 6 (6), 731-741 (2011).</li><li>Martinez Molina, D., Nordlund, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+cellular+thermal+shift+assay:+a+novel+biophysical+assay+for+in+situ+drug+target+engagement+and+mechanistic+biomarker+studies.">The cellular thermal shift assay: a novel biophysical assay for in situ drug target engagement and mechanistic biomarker studies.</a> Annual Review of Pharmacology and Toxicology. 56, 141-161 (2016).</li><li>Appelman, M. D., Chakraborty, A., Protzer, U., McKeating, J. A., van de Graaf, S. F. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=N-Glycosylation+of+the+Na+-taurocholate+cotransporting+polypeptide+(NTCP)+determines+its+trafficking+and+stability+and+is+required+for+hepatitis+B+virus+infection.">N-Glycosylation of the Na+-taurocholate cotransporting polypeptide (NTCP) determines its trafficking and stability and is required for hepatitis B virus infection.</a> PLoS One. 12 (1), 0170419 (2017).</li><li>Tsukuda, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+class+of+hepatitis+B+and+D+virus+entry+inhibitors,+proanthocyanidin+and+its+analogs,+that+directly+act+on+the+viral+large+surface+proteins.">A new class of hepatitis B and D virus entry inhibitors, proanthocyanidin and its analogs, that directly act on the viral large surface proteins.</a> Hepatology. 65 (4), 1104-1116 (2017).</li><li>Klumpp, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-resolution+crystal+structure+of+a+hepatitis+B+virus+replication+inhibitor+bound+to+the+viral+core+protein.">High-resolution crystal structure of a hepatitis B virus replication inhibitor bound to the viral core protein.</a> Proceedings of the National Academy of Sciences. 112 (49), 15196-15201 (2015).</li><li>Donkers, J. M., Appelman, M. D., van de Graaf, S. F. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanistic+insights+into+the+inhibition+of+NTCP+by+myrcludex+B.">Mechanistic insights into the inhibition of NTCP by myrcludex B.</a> JHEP Reports. 1 (4), 278-285 (2019).</li><li>Saso, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+strategy+to+identify+hepatitis+B+virus+entry+inhibitors+by+AlphaScreen+technology+targeting+the+envelope-receptor+interaction.">A new strategy to identify hepatitis B virus entry inhibitors by AlphaScreen technology targeting the envelope-receptor interaction.</a> Biochemical and Biophysical Research Communications. 501 (2), 374-379 (2018).</li><li>Baranauskiene, L., Kuo, T. C., Chen, W. Y., Matulis, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isothermal+titration+calorimetry+for+characterization+of+recombinant+proteins.">Isothermal titration calorimetry for characterization of recombinant proteins.</a> Current Opinion in Biotechnology. 55, 9-15 (2019).</li><li>Zhang, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structure-based+virtual+screening+protocol+for+in+silico+identification+of+potential+thyroid+disrupting+chemicals+targeting+transthyretin.">Structure-based virtual screening protocol for in silico identification of potential thyroid disrupting chemicals targeting transthyretin.</a> Environmental Science &amp; Technology. 50 (21), 11984-11993 (2016).</li><li>Duff, J. M. R., Grubbs, J., Howell, E. 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(55), e2796 (2011).</li><li>Du, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Insights+into+protein-ligand+interactions:+mechanisms,+models,+and+methods.">Insights into protein-ligand interactions: mechanisms, models, and methods.</a> International Journal of Molecular Sciences. 17 (2), 144 (2016).</li><li>Sureau, C., Salisse, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+conformational+heparan+sulfate+binding+site+essential+to+infectivity+overlaps+with+the+conserved+hepatitis+B+virus+A-determinant.">A conformational heparan sulfate binding site essential to infectivity overlaps with the conserved hepatitis B virus A-determinant.</a> Hepatology. 57 (3), 985-994 (2013).</li><li>Herrscher, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hepatitis+B+virus+entry+into+HepG2-NTCP+cells+requires+clathrin-mediated+endocytosis.">Hepatitis B virus entry into HepG2-NTCP cells requires clathrin-mediated endocytosis.</a> Cellular Microbiology. 22 (8), 13205 (2020).</li><li>Gripon, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Infection+of+a+human+hepatoma+cell+line+by+hepatitis+B+virus.">Infection of a human hepatoma cell line by hepatitis B virus.</a> Proceedings of the National Academy of Sciences. 99 (24), 15655-15660 (2002).</li><li>Mayati, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+polarization+of+human+hepatoma+HepaRG+cells+in+response+to+forskolin.">Functional polarization of human hepatoma HepaRG cells in response to forskolin.</a> Scientific Reports. 8 (1), 16115 (2018).</li><li>Sells, M. A., Chen, M. L., Acs, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Production+of+hepatitis+B+virus+particles+in+Hep+G2+cells+transfected+with+cloned+hepatitis+B+virus+DNA.">Production of hepatitis B virus particles in Hep G2 cells transfected with cloned hepatitis B virus DNA.</a> Proceedings of the National Academy of Sciences of the United States of America. 84 (4), 1005-1009 (1987).</li><li>Freyer, M. W., Lewis, E. 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HBV infection is initiated via low-affinity binding to heparan sulfate proteoglycans (HSPGs) on hepatocytes25, followed by the binding to NTCP with subsequent internalization through endocytosis26. As NTCP is a crucial receptor for HBV entry, targeting HBV entry can be clinically translated to diminish de novo infection, mother-to-child transmission (MTCT), and recurrence after liver transplantation. Interrupting viral entry would be a feasible alternative cure for...

Hepatitis B virus (HBV) infection is considered a life-threatening disease worldwide. Chronic HBV infection is laden with a risk of liver cirrhosis and hepatocellular carcinoma1. Current anti-HBV treatment focuses mostly on post viral entry using nucleos(t)ide analogs (NAs) and interferon-alpha (IFN-&#945;)2,3. The discovery of an HBV entry inhibitor, Myrcludex B, has identified a novel target for anti-HBV agents4. The combination of entry inhibitors and NAs in chronic HBV has significantly lessened the viral load compared to those targeting viral replication alone5,6. However, the classical hepatocyte model for the screening of HBV entry inhibitors is limited by low viral receptor levels (sodium taurocholate cotransporting polypeptide, NTCP). The overexpression of hNTCP in hepatoma cells (i.e., HepG2 and Huh7) improves HBV infectivity7,8. Nevertheless, these cell lines express low levels of phase I and II drug-metabolizing enzymes and exhibit genetic instability9. Hepatocyte models that can help target distinct mechanisms of candidate anti-HBV compounds such as previral entry, NTCP binding, and viral entry would expedite the identification and development of efficacious combination regimens. The study for anti-HBV activity of curcumin has elucidated the inhibition of viral entry as a new mechanism in addition to post viral entry interruption. This protocol details a host model for the screening of anti-HBV entry molecules10.
The goal of this method is to explore candidate anti-HBV compounds for viral entry inhibition, especially blocking NTCP binding and transport. As NTCP expression is a critical factor for HBV entry and infection, we optimized the hepatocyte maturation protocol to maximize NTCP levels11. In addition, this protocol can differentiate the inhibitory effect on HBV entry as inhibition of HBV attachment versus inhibition of internalization. The taurocholic acid (TCA) uptake assay was also modified using an ELISA-based method instead of a radioisotope to represent NTCP transport12,13. The receptor and ligand interaction was confirmed by their 3D structures14,15. The inhibition of NTCP function can be evaluated by measuring TCA uptake activity16. However, this technique did not provide direct evidence of NTCP binding to the candidate inhibitors. Therefore, the binding can be investigated using various techniques, such as surface plasmon resonance17, ELISA, fluorescence-based thermal shift assay (FTSA)18, FRET19, AlphaScreen, and various other methods20. Among these techniques, ITC is a goal standard in binding analysis because it can observe heat absorption or emission in almost every reaction21. The binding affinity (KD) of NTCP and candidate compounds was directly evaluated using ITC; these affinity values were more precise than those obtained using the in silico prediction model22.
This protocol covers techniques in hepatocyte maturation, HBV infection, and screening for HBV entry inhibitor. Briefly, a hepatocyte model was developed based on imHC and HepaRG cell lines. The cultured cells were differentiated into mature hepatocytes within 2 weeks. The upregulation of NTCP levels was detected using real-time PCR, western blot, and flow cytometry11. Hepatitis B virion (HBVcc) was produced and collected from HepG2.2.15. The differentiated imHC or HepaRG (d-imHC, d-HepaRG) was prophylactically treated with the anti-HBV candidates 2 h prior to the inoculation with HBV virion. The expected outcome of the experiment was the identification of the agents that decrease cellular HBV and infectivity. Anti-NTCP activity was evaluated using the TCA uptake assay. NTCP activity could be suppressed by the agents that specifically bound NTCP. The ITC technique was employed to investigate the feasibility of interactive binding that could predict inhibitors and their target proteins, determining the binding affinity (KD) of the ligand for the receptor via non-covalent interactions of the biomolecular complex23,24. For instance, KD&#160;&#8805; 1 &#215; 103 mM represents weak binding, KD&#160;&#8805; 1 &#215; 106 &#181;M represents moderate binding, and KD&#160;&#8804; 1 &#215; 109 nM represents strong binding. The &#916;G is directly correlated with binding interactions. In particular, a reaction with negative &#916;G is an exergonic reaction, indicating that binding is a spontaneous process. A reaction with a negative &#916;H indicates that the binding processes depend on hydrogen bonding and Van der Waals forces. Both TCA uptake and ITC data could be used to screen for anti-HBV entry agents. The outcomes of these protocols can provide a foundation for not only anti-HBV screening but also the interaction with NTCP as assessed through binding affinity and transport function. This paper describes host cell preparation and characterization, experimental design, and evaluation of the anti-HBV entry together with the NTCP binding affinity.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Cell lines</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>HepaRG Cells, Cryopreserved</td>
			<td>Thermo Fisher Scientific</td>
			<td>HPRGC10</td>
			<td></td>
		</tr>
		<tr>
			<td>Hep-G2/2.2.15 Human Hepatoblastoma Cell Line</td>
			<td>Merck</td>
			<td>SCC249</td>
			<td></td>
		</tr>
		<tr>
			<td>Reagents</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>4% Paraformadehyde Phosphate Buffer Solution</td>
			<td>FUJIFLIM Wako chemical</td>
			<td>163-20145</td>
			<td></td>
		</tr>
		<tr>
			<td>BD Perm/Wash buffer</td>
			<td>BD Biosciences</td>
			<td>554723</td>
			<td>Perm/Wash buffer</td>
		</tr>
		<tr>
			<td>Cyclosporin A</td>
			<td>abcam</td>
			<td>59865-13-3</td>
			<td></td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>Invitrogen</td>
			<td>15575-038</td>
			<td>8 mM</td>
		</tr>
		<tr>
			<td>G 418 disulfate salt</td>
			<td>Merck</td>
			<td>108321-42-2</td>
			<td></td>
		</tr>
		<tr>
			<td>Halt Protease Inhibitor Cocktail&#160; EDTA-free (100x)</td>
			<td>Thermo Scientific</td>
			<td>78425</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Merck</td>
			<td>7365-45-9</td>
			<td></td>
		</tr>
		<tr>
			<td>illustraTM RNAspin Mini RNA isolation kits</td>
			<td>GE Healthcare</td>
			<td>25-0500-71</td>
			<td></td>
		</tr>
		<tr>
			<td>illustra RNAspin Mini RNA Isolation Kit</td>
			<td>GE Healthcare</td>
			<td>25-0500-71</td>
			<td></td>
		</tr>
		<tr>
			<td>ImProm-II Reverse Transcription System</td>
			<td>Promega</td>
			<td>A3800</td>
			<td></td>
		</tr>
		<tr>
			<td>KAPA SYBR FAST qPCR Kit</td>
			<td>Kapa Biosystems</td>
			<td>KK4600</td>
			<td></td>
		</tr>
		<tr>
			<td>Lenti-X Concentrator</td>
			<td>Takara bio</td>
			<td>PT4421-2</td>
			<td>concentrator</td>
		</tr>
		<tr>
			<td>Luminata crescendo Western HRP substrate</td>
			<td>Merck</td>
			<td>WBLUR0100</td>
			<td></td>
		</tr>
		<tr>
			<td>Master Mix (2x) Universal</td>
			<td>Kapa Biosystems</td>
			<td>KK4600</td>
			<td></td>
		</tr>
		<tr>
			<td>Nucleospin DNA extraction kit</td>
			<td>macherey-nagel</td>
			<td>1806/003</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate buffered saline</td>
			<td>Merck</td>
			<td>P3813</td>
			<td></td>
		</tr>
		<tr>
			<td>Polyethylene glycol 8000</td>
			<td>Merck</td>
			<td>25322-68-3</td>
			<td></td>
		</tr>
		<tr>
			<td>ProLong Gold Antifade Mountant</td>
			<td>Thermo scientific</td>
			<td>P36930</td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant NTCP</td>
			<td>Cloud-Clone</td>
			<td>RPE421Hu02</td>
			<td></td>
		</tr>
		<tr>
			<td>RIPA Lysis Buffer (10x)</td>
			<td>Merck</td>
			<td>20-188</td>
			<td></td>
		</tr>
		<tr>
			<td>TCA</td>
			<td>Sigma</td>
			<td>345909-26-4</td>
			<td></td>
		</tr>
		<tr>
			<td>TCA Elisa kit</td>
			<td>Mybiosource</td>
			<td>MB2033685</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Merck</td>
			<td>9036-19-5</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin-EDTA</td>
			<td>Gibco</td>
			<td>25200072</td>
			<td>Dilute to 0.125%</td>
		</tr>
		<tr>
			<td>Antibodies</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>&#160;&#160;&#160; Anti-NTCP1 antibody</td>
			<td>Abcam</td>
			<td>ab131084</td>
			<td>1:100 dilution</td>
		</tr>
		<tr>
			<td>&#160;&#160;&#160; Anti-GAPDH antibody</td>
			<td>Thermo Fisher Scientific</td>
			<td>AM4300</td>
			<td>1:200,000 dilution</td>
		</tr>
		<tr>
			<td>&#160;&#160; HRP-conjugated goat anti-rabbit antibody</td>
			<td>Abcam</td>
			<td>ab205718</td>
			<td>1:10,000 dilution</td>
		</tr>
		<tr>
			<td>&#160;&#160; HRP goat anti-mouse secondary antibody</td>
			<td>Abcam</td>
			<td>ab97023</td>
			<td>1:10,000 dilution</td>
		</tr>
		<tr>
			<td>&#160;&#160; Goat anti-Rabbit IgG Secondary Antibody, Alexa Fluor 488</td>
			<td>Invitrogen</td>
			<td>A-11008</td>
			<td>1:500 dilution</td>
		</tr>
		<tr>
			<td>Reagent composition</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>1&#176; Antibody dilution buffer</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>&#160;&#160;&#160;&#160; 1x TBST</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>&#160;&#160;&#160;&#160; 3% BSA</td>
			<td>Sigma</td>
			<td>A7906-100G</td>
			<td>Working concentration: 3%</td>
		</tr>
		<tr>
			<td>&#160;&#160;&#160;&#160; Sodium azide</td>
			<td>Sigma</td>
			<td>199931</td>
			<td>Working concentration: 0.05%</td>
		</tr>
		<tr>
			<td>Hepatocyte Growth Medium</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>&#160;&#160;&#160;&#160;&#160; DME/F12</td>
			<td>Gibco</td>
			<td>12400-024</td>
			<td></td>
		</tr>
		<tr>
			<td>&#160;&#160;&#160;&#160;&#160; 10% FBS</td>
			<td>Sigma Aldrich</td>
			<td>F7524</td>
			<td></td>
		</tr>
		<tr>
			<td>&#160;&#160;&#160;&#160;&#160; 1% Pen/Strep</td>
			<td>HyClon</td>
			<td>SV30010</td>
			<td></td>
		</tr>
		<tr>
			<td>&#160;&#160;&#160;&#160;&#160; 1% GlutaMAX</td>
			<td>Gibco</td>
			<td>35050-061</td>
			<td></td>
		</tr>
		<tr>
			<td>Hepatic maturation medium</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>&#160;&#160;&#160;&#160;&#160; Williams&#8217; E medium</td>
			<td>Sigma Aldrich</td>
			<td>W4125-1L</td>
			<td></td>
		</tr>
		<tr>
			<td>&#160;&#160;&#160;&#160;&#160; 10% FBS</td>
			<td>Sigma Aldrich</td>
			<td>F7524</td>
			<td></td>
		</tr>
		<tr>
			<td>&#160;&#160;&#160;&#160;&#160; 1% Pen/Strep</td>
			<td>HyClon</td>
			<td>SV30010</td>
			<td></td>
		</tr>
		<tr>
			<td>&#160;&#160;&#160;&#160;&#160; 1% GlutaMAX</td>
			<td>Gibco</td>
			<td>35050-061</td>
			<td></td>
		</tr>
		<tr>
			<td>&#160;&#160;&#160;&#160;&#160; 5 &#181;g/mL&#160; Insulin</td>
			<td>Sigma Aldrich</td>
			<td>91077C-100MG</td>
			<td></td>
		</tr>
		<tr>
			<td>&#160;&#160;&#160;&#160;&#160; 50 &#181;M hydrocotisone</td>
			<td>Sigma Aldrich</td>
			<td>H0888-1g</td>
			<td></td>
		</tr>
		<tr>
			<td>&#160;&#160;&#160;&#160; 2% DMSO</td>
			<td>PanReac AppliChem</td>
			<td>A3672-250ml</td>
			<td></td>
		</tr>
		<tr>
			<td>IF Blocking solution</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>&#160;&#160;&#160;&#160; 1x PBS</td>
			<td>Gibco</td>
			<td>21300-058</td>
			<td></td>
		</tr>
		<tr>
			<td>&#160;&#160;&#160;&#160; 3% BSA</td>
			<td>Sigma</td>
			<td>A7906-100G</td>
			<td>Working concentration: 3%</td>
		</tr>
		<tr>
			<td>&#160;&#160;&#160;&#160; 0.2% Triton X-100</td>
			<td>Sigma</td>
			<td>T8787</td>
			<td>Working concentration: 0.2%</td>
		</tr>
		<tr>
			<td>RIPA Lysis Buffer Solution</td>
			<td>Merck</td>
			<td>20-188</td>
			<td>Final concentration: 1X</td>
		</tr>
		<tr>
			<td>&#160;&#160;&#160;&#160; Protease Inhibitor Cocktail</td>
			<td>Thermo Scientific</td>
			<td>78425</td>
			<td>Final concentration: 1X</td>
		</tr>
		<tr>
			<td>&#160;&#160;&#160;&#160;&#160;&#160; Na3VO4</td>
			<td></td>
			<td></td>
			<td>Final concentration: 1 mM</td>
		</tr>
		<tr>
			<td>&#160;&#160;&#160;&#160;&#160;&#160; PMSF</td>
			<td></td>
			<td></td>
			<td>Final concentration: 1 mM</td>
		</tr>
		<tr>
			<td>&#160;&#160;&#160;&#160;&#160;&#160; NaF</td>
			<td></td>
			<td></td>
			<td>Final concentration: 10 mM</td>
		</tr>
		<tr>
			<td>Western blot reagent</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>&#160;&#160;&#160;&#160; 10x Tris-buffered saline (TBS)</td>
			<td>Bio-Rad</td>
			<td>170-6435</td>
			<td>Final concentration: 1X</td>
		</tr>
		<tr>
			<td>&#160;&#160;&#160;&#160; Tween 20</td>
			<td>Merck</td>
			<td>9005-64-5</td>
			<td></td>
		</tr>
		<tr>
			<td>&#160;&#160;&#160;&#160; 1x TBST</td>
			<td></td>
			<td></td>
			<td>0.1% Tween 20</td>
		</tr>
		<tr>
			<td>&#160;&#160;&#160;&#160; 1x PBS</td>
			<td>Gibco</td>
			<td>21300-058</td>
			<td></td>
		</tr>
		<tr>
			<td>&#160;&#160;&#160;&#160; Pierce BCA Protein Assay Kit</td>
			<td>Thermo Fisher Scientific</td>
			<td>A53225</td>
			<td></td>
		</tr>
		<tr>
			<td>&#160;&#160;&#160;&#160; Polyacrylamide gel</td>
			<td>Bio-Rad</td>
			<td>161-0183</td>
			<td></td>
		</tr>
		<tr>
			<td>&#160;&#160;&#160;&#160; Ammonium Persulfate (APS)</td>
			<td>Bio-Rad</td>
			<td>161-0700</td>
			<td>Final concentration: 0.05%</td>
		</tr>
		<tr>
			<td>&#160;&#160;&#160; TEMED</td>
			<td>Bio-Rad</td>
			<td>161-0800</td>
			<td>Stacker gel: 0.1%, Resolver gel: 0.05%</td>
		</tr>
		<tr>
			<td>&#160;&#160;&#160; 2x Laemmli Sample Buffer</td>
			<td>Bio-Rad</td>
			<td>161-0737</td>
			<td>Final concentration: 1X</td>
		</tr>
		<tr>
			<td>&#160;&#160;&#160; Precision Plus Protein Dual Color Standards</td>
			<td>Bio-Rad</td>
			<td>161-0374</td>
			<td></td>
		</tr>
		<tr>
			<td>WB Blocking solution/ 2&#176; Antibody dilution buffer</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>&#160;&#160;&#160;&#160; 1x TBST</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>&#160;&#160;&#160;&#160; 5% Skim milk (nonfat dry milk)</td>
			<td>Bio-Rad</td>
			<td>170-6404</td>
			<td>Working concentration: 5%</td>
		</tr>
		<tr>
			<td>1x Running buffer 1 L</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>&#160;&#160;&#160;&#160;&#160; 10x Tris-buffered saline (TBS)</td>
			<td>Bio-Rad</td>
			<td>170-6435</td>
			<td>Final concentration: 1X</td>
		</tr>
		<tr>
			<td>&#160;&#160;&#160;&#160; Glycine</td>
			<td>Sigma</td>
			<td>G8898</td>
			<td>14.4 g</td>
		</tr>
		<tr>
			<td>&#160;&#160;&#160;&#160; SDS</td>
			<td>Merck</td>
			<td>7910</td>
			<td>Working concentration: 0.1%</td>
		</tr>
		<tr>
			<td>Blot transfer buffer 500 mL</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>&#160;&#160;&#160;&#160;&#160; 10x Tris-buffered saline (TBS)</td>
			<td>Bio-Rad</td>
			<td>170-6435</td>
			<td>Final concentration: 1X</td>
		</tr>
		<tr>
			<td>&#160;&#160;&#160;&#160; Glycine</td>
			<td>Sigma</td>
			<td>G8898</td>
			<td>7.2 g</td>
		</tr>
		<tr>
			<td>&#160;&#160;&#160;&#160; Methanol</td>
			<td>Merck</td>
			<td>106009</td>
			<td>100 mL</td>
		</tr>
		<tr>
			<td>Mild stripping solution 1 L</td>
			<td></td>
			<td></td>
			<td>Adjust pH to 2.2</td>
		</tr>
		<tr>
			<td>&#160;&#160;&#160; Glycine</td>
			<td>Sigma</td>
			<td>G8898</td>
			<td>15 g</td>
		</tr>
		<tr>
			<td>&#160;&#160;&#160;&#160; SDS</td>
			<td>Merck</td>
			<td>7910</td>
			<td>1 g</td>
		</tr>
		<tr>
			<td>&#160;&#160;&#160;&#160; Tween 20</td>
			<td>Merck</td>
			<td>9005-64-5</td>
			<td>10 mL</td>
		</tr>
		<tr>
			<td>Equipments</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>15 mL centrifuge tube</td>
			<td>Corning</td>
			<td>430052</td>
			<td></td>
		</tr>
		<tr>
			<td>50 mL centrifuge tube</td>
			<td>Corning</td>
			<td>430291</td>
			<td></td>
		</tr>
		<tr>
			<td>Airstream Class II</td>
			<td>Esco</td>
			<td>2010621</td>
			<td>Biological safety cabinet</td>
		</tr>
		<tr>
			<td>CelCulture CO2 Incubator</td>
			<td>Esco</td>
			<td>2170002</td>
			<td>Humidified tissue culture incubator</td>
		</tr>
		<tr>
			<td>CFX96 Touch Real-Time PCR Detector</td>
			<td>Bio-Rad</td>
			<td>1855196</td>
			<td></td>
		</tr>
		<tr>
			<td>FACSVerse Flow Cytometer</td>
			<td>BD Biosciences</td>
			<td>651154</td>
			<td></td>
		</tr>
		<tr>
			<td>Graduated pipettes (10 mL)</td>
			<td>Jet Biofil</td>
			<td>GSP010010</td>
			<td></td>
		</tr>
		<tr>
			<td>Graduated pipettes (5 mL)</td>
			<td>Jet Biofil</td>
			<td>GSP010005</td>
			<td></td>
		</tr>
		<tr>
			<td>MicroCal PEAQ-ITC</td>
			<td>Malvern</td>
			<td></td>
			<td>Isothermal titration calorimeters</td>
		</tr>
		<tr>
			<td>Mini PROTEAN Tetra Cell</td>
			<td>Bio-Rad</td>
			<td>1658004</td>
			<td>Electrophoresis chamber</td>
		</tr>
		<tr>
			<td>Mini Trans-blot absorbent filter paper</td>
			<td>Bio-Rad</td>
			<td>1703932</td>
			<td></td>
		</tr>
		<tr>
			<td>Omega Lum G Imaging System</td>
			<td>Aplegen</td>
			<td>8418-10-0005</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipette controller</td>
			<td>Eppendorf</td>
			<td>4430000.018</td>
			<td>Easypet 3</td>
		</tr>
		<tr>
			<td>PowerPac HC</td>
			<td>Bio-Rad</td>
			<td>1645052</td>
			<td>Power supply</td>
		</tr>
		<tr>
			<td>PVDF membrane</td>
			<td>Merck</td>
			<td>IPVH00010</td>
			<td></td>
		</tr>
		<tr>
			<td>T-75 A91:D106flask</td>
			<td>Corning</td>
			<td>431464U</td>
			<td></td>
		</tr>
		<tr>
			<td>Trans-Blot SD Semi-Dry Transfer Cell</td>
			<td>Bio-Rad</td>
			<td>1703940</td>
			<td>Semi-dry transfer cell</td>
		</tr>
		<tr>
			<td>Ultrasonic processor (Vibra-Cell VCX 130)</td>
			<td>Sonics &#38; Materials</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Versati Tabletop Refrigerated Centrifuge</td>
			<td>Esco</td>
			<td>T1000R</td>
			<td>Centrifuge with swinging bucket rotar</td>
		</tr>
	</tbody>
</table>

a competent hepatocyte model examining hepatitis b virus entry through sodium taurocholate cotransporting polypeptide as a therapeutic target

Hepatitis B virus (HBV) infection has been considered a crucial risk factor for hepatocellular carcinoma. Current treatment can only lessen the viral load but not result in complete remission. An efficient hepatocyte model for HBV infection would offer a true-to-life viral life cycle that would be crucial for the screening of therapeutic agents. Most available anti-HBV agents target lifecycle stages post viral entry but not before viral entry. This protocol details the generation of a competent hepatocyte model capable of screening for therapeutic agents targeting pre-viral entry and post viral entry lifecycle stages. This includes the targeting of sodium taurocholate cotransporting polypeptide (NTCP) binding, cccDNA formation, transcription, and viral assembly based on imHC or HepaRG as host cells. Here, the HBV entry inhibition assay used curcumin to inhibit HBV binding and transporting functions via NTCP. The inhibitors were evaluated for binding affinity (KD) with NTCP using isothermal titration calorimetry (ITC)-a universal tool for HBV drug screening based on thermodynamic parameters.

Hepatic maturation features were observed, including binucleated cells and polygonal-shaped morphology (Figure 1), especially in the differentiated stage of imHC (Figure 1A). A large increase in NTCP expression was measured in d-HepaRG and d-imHC at 7-fold and 40-fold, respectively (Figure 1B). The highly glycosylated form of NTCP, postulated to confer susceptibility to HBV entry, was detected more in d-imHC than in d-HepaRG (<stron...

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A Competent Hepatocyte Model Examining Hepatitis B Virus Entry through Sodium Taurocholate Cotransporting Polypeptide as a Therapeutic Target

We present a protocol to screen anti-hepatitis B virus (HBV) compounds targeting pre- and post-viral entry lifecycle stages, using isothermal titration calorimetry to measure binding affinity (KD) with host sodium taurocholate cotransporting polypeptide. Antiviral efficacy was determined through the suppression of viral lifecycle markers (cccDNA formation, transcription, and viral assembly).

Hepatitis B virus (HBV) infection has been considered a crucial risk factor for hepatocellular carcinoma. Current treatment can only lessen the viral load ...

This protocol is designed to verify if a candidate anti HBV compound inhibits viral entry especially by blocking NTCP binding. This technique is intended to investigate if HBV entry or an initial step of infection could be interrupted by any candidate compound. Demonstrating the procedure will be Piyanoot Thongsri, a PSP student from my laboratory.To begin the measurement of the sodium taurocholate co-transporting polypeptide, or NTCP, incubate the mature hepatocytes with eight mill or more ethylenediaminetetraacetic acid or EDTA for 15 minutes. Collect cell pellets and fix the hepatocytes by adding 300 microliters of 3.7%paraformaldehyde in PBS for 15 minutes. Then transfer the cell suspension into a 1.5 milliliter microcentrifuge tube to centrifuge them at 300 G and 25 degrees Celsius for 10 minutes.Wash the cells twice using 700 microliters of PBS containing 1%fetal bovine serum or FBS in centrifuge for 10 minutes as described before. Then permeabilize the cells with 300 microliters of 0.05%Triton X 100 in PBS at room temperature for 20 minutes. After the centrifugation give three washes of one minute each using 700 microliters of PBS containing 1%FBS.Incubate the cells for 30 minutes at four degrees Celsius with the primary antibody against NTCP in a one to 100 ratio, followed by three washes with perm wash buffer, and centrifugation. Stain the cells with a secondary antibody conjugated to Alexa Fluor 488 for 30 minutes at four degrees Celsius and repeat three washes of perm wash buffer for one minute each. After centrifugation at 300 G, resuspend the cell pellet with 200 microliters of PBS containing 1%FBS.In the software, select the measurement parameters for flow cytometric analysis, including FSC, SSC, FITC, or PE.Set auto compensation adjustment and include the compensation controls as explained in the manuscript. Next, run the unstained sample with a medium fluidic flow rate of 60 microliters per minute. Adjust the forward scatter and side scatter threshold until they cover the cell population of interest.Mark the gate region in this area and apply to all compensation controls. Set the photo multiplier tube or PMT fluorescence using the unstained control and adjust the PMT voltage of FITC or PE in the negative quadrant. Run the positive stained sample and adjust FITC or PE in the positive quadrant scale.Run the unstained, FITC stained or PE controls, calculate the compensation, and apply it to the sample settings. Next, run the hepatocyte sample to measure the NTCP level. Next, start analyzing the stained hepatocytes by serially selecting on BD FACSuite windows, the control tube, and then data sources tab.Wait for the raw data to appear. Next, in the worksheet tab on the right hand side click on the ellipse gate button, select the cell population and SSC and the FSC dot plot using the mouse cursor. Then wait for the circle P1 to appear.Click on the interval gate button and mark the region in the fluorescein isothiocyanate stained histogram. Starting from the highest fluorescence intensity value to the right hand side. The line P2 appears on the screen.Click on the experiment tube and observe that the data in the worksheet tab changes. Click on the statistics button and then on the empty space and the worksheet. Then, observe the statistical values of the sodium taurocholate co-transporting polypeptide population.Aspirate the medium from the 100%confluent MHC well plates and add four micromolar cyclosporine A diluted with William's E Medium for two hours. Next, aspirate the candidate hepatitis B virus entry inhibitor to replace it with one milliliter of Williams E Medium containing 4%polyethylene glycol. Then, incubate the culture medium with hepatitis B virus particles at a multiplicity of infection of 100 per well for 18 hours at 37 degrees Celsius.Then, discard the supernatant at two milliliters of cold PBS and swirl gently to rinse off the old medium with unbound hepatitis B virus. After culturing the cells with two milliliters of complete William's E Medium for seven days wash the cells with PBS and fix them with 3.7%paraform aldehyde and PBS for 15 minutes at room temperature. Sequentially incubate the infected cells with IF blocking solution for 60 minutes at room temperature in the primary antibody at one to 200 dilution in the blocking solution overnight at four degrees Celsius.Then give three washes of one minute each using washing solution before incubating the cells with the secondary antibody in one to 500 dilution for one hour at room temperature. After washing thrice, mount the sample with an anti fade mounting medium with DAPI and cover it with a glass cover slip. Using a fluorescence microscope equipped having DAPI and PE filters detect the fluorescence signal with a 20 x objective lens to determine the anti HBV entry activity of the candidate compounds.For the cell binding assay, prepare the cells as demonstrated before and incubate them with hepatitis B virus particles at four degrees Celsius for two hours. After discarding the medium, rinse the cells with two milliliters of cold PBS with a gentle swirl. Then harvest the infected cells using a cell scraper and disrupt the cells with lysis buffer.Transfer the cell lysate to a collection tube to extract the total DNA with polymerase chain reaction using the temperature conditions described in the manuscript. After preparing the cells as demonstrated before, incubate the hepatocytes with the sodium taurocholic acid solution for 15 minutes at room temperature, aspirate the solution and wash the cells thrice with cold HEPES. Next, add 300 to 500 microliters of cold HEPES to harvest the cells with cell scrapers on ice.Homogenize the cells by ultrasonication at 20 kilohertz for 20 seconds, and incubate on ice for five minutes. After centrifugation at 10, 000 G, collect the supernatant and evaluate intracellular taurocholic acid concentration using a taurocholic acid ELISA assay kit. To wash the cells and syringe in the isothermal titration calorimetry or ITC instrument, launch the ITC software.Under the run experiment highlight tab, select micro cal method for 19 injection dot ITCM and click open. When a new window opens, click on the clean tab and in the cleaning method window select the wash button for cell cleaning method and the rinse button for syringe cleaning method. Click on next and follow the onscreen instructions.After loading the sample into the ITC, click on the load button present under the run experiment tab. Then click next and follow the video instructions until this loading step is completed. Using a syringe, fill the reference cell with solution buffer in the sample cell with 15 micromolar NTCP in the buffer.Remove the excess solution, then fill the syringe with a 150 Micromolar Curcumin Ligand solution, avoiding air bubbles. Run the sample by clicking on the run button under the run experiment tab. The experimental information and settings will be displayed on the left hand side.Enter the ligand protein concentrations and temperature details. Click on start to perform the injection process. Wait for 1.4 hours for the protein ligand interaction to complete.Once the injection is complete select the highlighted analysis button to analyze the raw data using the analysis software. Then, click on overview to display the raw data and choose the fitting model as one set of binding site. Hepatic maturation features were observed including binucleated cells and polygonal shaped morphology, especially in the differentiated stage of MHC.A large increase in NTCP expression was observed in differentiated HepaRG and differentiated MHC. The highly glycosylated form of NTCP was detected more in differentiated MHC than in differentiated HepaRG. The NTCP levels were higher in differentiated MHC cells than in the undifferentiated cells.Virus immunofluorescence staining evaluated the anti HBV entry activities on day seven post-infection and the level of virus binding on the cell surface receptor was evaluated by real-time polymerase chain reaction. Taurocholic acid uptake analysis suggested that the punitive inhibitory activity of candidate compounds decreased Hepatitis B virus binding through NTCP. The colorimetric alteration was detected with continuous injections to the sample cell.A standard non-linear least squares regression was plotted based on one binding site fitted well to the data. The solid line indicated the best fit to the experimental values For SBB binding as a and that cells and hepatitis B viral particle are incubated at four degrees Celsius. Taurocholic acid update can be evaluated using the radioactive technique.The level of radioactive taurocholic acid can be quantified using a liquid simulation counter. This technique is simple and quick to screen for viral entry in inquiry activity of any antiviral regimen that would help prevent either infection or reinfection.

a-competent-hepatocyte-model-examining-hepatitis-b-virus-entry

Research

JoVE Journal

Biochemistry

Directed Protein Packaging within Outer Membrane Vesicles from Escherichia coli: Design, Production and Purification

An increasing interest in applying synthetic biology techniques to program outer membrane vesicles (OMV) are leading to some very interesting and unique applications for OMV where traditional nanoparticles are proving too difficult to synthesize. To date, all Gram-negative bacteria have been shown to produce OMV demonstrating packaging of a variety of cargo that includes small molecules, peptides, proteins and genetic material. Based on their diverse cargo, OMV are implicated in many biological processes ranging from cell-cell communication to gene transfer and delivery of virulence factors depending upon which bacteria are producing the OMV. Only recently have bacterial OMV become accessible for use across a wide range of applications through the development of techniques to control and direct packaging of recombinant proteins into OMV. This protocol describes a method for the production, purification, and use of enzyme packaged OMV providing for improved overall production of recombinant enzyme, increased vesiculation, and enhanced enzyme stability. Successful utilization of this protocol will result in the creation of a bacterial strain that simultaneously produces a recombinant protein and directs it for OMV encapsulation through creating a synthetic linkage between the recombinant protein and an outer membrane anchor protein. This protocol also details methods for isolating OMV from bacterial cultures as well as proper handling techniques and things to consider when adapting this protocol for use for other unique applications such as: pharmaceutical drug delivery, medical diagnostics, and environmental remediation.

Directed Protein Packaging within Outer Membrane Vesicles from Escherichia coli: Design, Production and Purification

A protocol for the production, purification, and use of enzyme packaged outer membrane vesicles (OMV) providing for enhanced enzyme stability for implementation across diverse applications is presented.

An increasing interest in applying synthetic biology techniques to program outer membrane vesicles (OMV) are leading to some very interesting and unique ...

Fluorescence Anisotropy as a Tool to Study Protein-protein Interactions

Protein-protein interactions play an essential role in the function of a living organism. Once an interaction has been identified and validated it is necessary to characterize it at the structural and mechanistic level. Several biochemical and biophysical methods exist for such purpose. Among them, fluorescence anisotropy is a powerful technique particularly used when the fluorescence intensity of a fluorophore-labeled protein remains constant upon protein-protein interaction. In this technique, a fluorophore-labeled protein is excited with vertically polarized light of an appropriate wavelength that selectively excites a subset of the fluorophores according to their relative orientation with the incoming beam. The resulting emission also has a directionality whose relationship in the vertical and horizontal planes defines anisotropy (r) as follows: r=(IVV-IVH)/(IVV+2IVH), where IVV and IVH are the fluorescence intensities of the vertical and horizontal components, respectively. Fluorescence anisotropy is sensitive to the rotational diffusion of a fluorophore, namely the apparent molecular size of a fluorophore attached to a protein, which is altered upon protein-protein interaction. In the present text, the use of fluorescence anisotropy as a tool to study protein-protein interactions was exemplified to address the binding between the protein mutated in the Shwachman-Diamond Syndrome (SBDS) and the Elongation factor like-1 GTPase (EFL1). Conventionally, labeling of a protein with a fluorophore is carried out on the thiol groups (cysteine) or in the amino groups (the N-terminal amine or lysine) of the protein. However, SBDS possesses several cysteines and lysines that did not allow site directed labeling of it. As an alternative technique, the dye 4&#39;,5&#39;-bis(1,3,2 dithioarsolan-2-yl) fluorescein was used to specifically label a tetracysteine motif, Cys-Cys-Pro-Gly-Cys-Cys, genetically engineered in the C-terminus of the recombinant SBDS protein. Fitting of the experimental data provided quantitative and mechanistic information on the binding mode between these proteins.

Protein interactions are at the heart of a cell&#39;s function. Calorimetric and spectroscopic techniques are commonly used to characterize them. Here we describe fluorescence anisotropy as a tool to study the interaction between the protein mutated in the Shwachman-Diamond Syndrome (SBDS) and the Elongation factor-like 1 GTPase (EFL1).

Protein-protein interactions play an essential role in the function of a living organism. Once an interaction has been identified and validated it is ...

Examining Proteasome Assembly with Recombinant Archaeal Proteasomes and Nondenaturing PAGE:  The Case for a Combined Approach

Proteasomes are found in all domains of life. They provide the major route of intracellular protein degradation in eukaryotes, though their assembly is not completely understood. All proteasomes contain a structurally conserved core particle (CP), or 20S proteasome, containing two heptameric&#160;&#946; subunit rings sandwiched between two heptameric&#160;&#945; subunit rings. Archaeal 20S proteasomes are compositionally simpler compared to their eukaryotic counterparts, yet they both share a common assembly mechanism. Consequently, archaeal 20S proteasomes continue to be important models for eukaryotic proteasome assembly. Specifically, recombinant expression of archaeal 20S proteasomes coupled with nondenaturing polyacrylamide gel electrophoresis (PAGE) has yielded many important insights into proteasome biogenesis. Here, we discuss a means to improve upon the usual strategy of coexpression of archaeal proteasome &#945; and &#946; subunits prior to nondenaturing PAGE. We demonstrate that although rapid and efficient, a coexpression approach alone can miss key assembly intermediates. In the case of the proteasome, coexpression may not allow detection of the half-proteasome, an intermediate containing one complete &#945;-ring and one complete &#946;-ring. However, this intermediate is readily detected via lysate mixing. We suggest that combining coexpression with lysate mixing yields an approach that is more thorough in analyzing assembly, yet remains labor nonintensive. This approach may be useful for the study of other recombinant multiprotein complexes.

This protocol uses both subunit coexpression and postlysis subunit mixing for a more thorough examination of recombinant proteasome assembly.

Proteasomes are found in all domains of life. They provide the major route of intracellular protein degradation in eukaryotes, though their assembly is ...

Laboratory Scale Production and Purification of a Therapeutic Antibody

Ensuring the successful production of a therapeutic antibody begins early on in the development process. The first stage is vector expression of the antibody genes followed by stable transfection into a suitable cell line. The stable clones are subjected to screening in order to select those clones with desired production and growth characteristics. This is a critical albeit time-consuming step in the process. This protocol considers vector selection and sourcing of antibody sequences for the expression of a therapeutic antibody. The methods describe preparation of vector DNA for stable transfection of a suspension variant of human embryonic kidney 293 (HEK-293) cell line, using polyethylenimine (PEI). The cells are transfected as adherent cells in serum-containing media to maximize transfection efficiency, and afterwards adapted to serum-free conditions. Large scale production, setup as batch overgrow cultures is used to yield antibody protein that is purified by affinity chromatography using an automated fast protein liquid chromatography (FPLC) instrument. The antibody yields produced by this method can provide sufficient protein to begin initial characterization of the antibody. This may include in vitro assay development or physicochemical characterization to aid in the time-consuming task of clonal screening for lead candidates. This method can be transferable to the development of an expression system for the production of biosimilar antibodies.

This protocol describes the production of a therapeutic antibody in a mammalian expression system. The methods described include preparation of vector DNA, stable transfection and serum-free adaptation of a human embryonic kidney 293 cell line, set up of large scale cultures and purification using affinity chromatography.

Ensuring the successful production of a therapeutic antibody begins early on in the development process. The first stage is vector expression of the ...

Differential Scanning Calorimetry &#8212; A Method for Assessing the Thermal Stability and Conformation of Protein Antigen

Differential scanning calorimetry (DSC) is an analytical technique that measures the molar heat capacity of samples as a function of temperature. In the case of protein samples, DSC profiles provide information about thermal stability, and to some extent serves as a structural &#8220;fingerprint&#8221; that can be used to assess structural conformation. It is performed using a differential scanning calorimeter that measures the thermal transition temperature (melting temperature; Tm) and the energy required to disrupt the interactions stabilizing the tertiary structure (enthalpy; &#8710;H) of proteins. Comparisons are made between formulations as well as production lots, and differences in derived values indicate differences in thermal stability and structural conformation. Data illustrating the use of DSC in an industrial setting for stability studies as well as monitoring key manufacturing steps are provided as proof of the effectiveness of this protocol. In comparison to other methods for assessing the thermal stability of protein conformations, DSC is cost-effective, requires few sample preparation steps, and also provides a complete thermodynamic profile of the protein unfolding process.

Differential Scanning Calorimetry &amp;#8212; A Method for Assessing the Thermal Stability and Conformation of Protein Antigen

Differential scanning calorimetry measures the thermal transition temperature(s) and total heat energy required to denature a protein. Results obtained are used to assess the thermal stability of protein antigens in vaccine formulations.

Differential scanning calorimetry (DSC) is an analytical technique that measures the molar heat capacity of samples as a function of temperature. In the ...

Detergent-free Ultrafast Reconstitution of Membrane Proteins into Lipid Bilayers Using Fusogenic Complementary-charged Proteoliposomes.

Detergents are indispensable for delivery of membrane proteins into 30-100 nm small unilamellar vesicles, while more complex, larger model lipid bilayers are less compatible with detergents.
Here we describe a strategy for bypassing this fundamental limitation using fusogenic oppositely charged liposomes bearing a membrane protein of interest. Fusion between such vesicles occurs within 5 min in a low ionic strength buffer. Positively charged fusogenic liposomes can be used as simple shuttle vectors for detergent-free delivery of membrane proteins into biomimetic target lipid bilayers, which are negatively charged. We also show how to reconstitute membrane proteins into fusogenic proteoliposomes with a fast 30-min protocol.
Combining these two approaches, we demonstrate a fast assembly of an electron transport chain consisting of two membrane proteins from E. coli, a primary proton pump bo3-oxidase and F1Fo ATP synthase, in membranes of vesicles of various sizes, ranging from 0.1 to &#62;10 microns, as well as ATP production by this chain.

Here we present two ultrafast protocols for reconstitution of membrane proteins into fusogenic proteoliposomes, and fusion of such proteoliposomes with target lipid bilayers for detergent-free delivery of these membrane proteins into the postfusion bilayer. The combination of these approaches enables fast and easily controlled assembly of complex multi-component membrane systems.

Detergents are indispensable for delivery of membrane proteins into 30-100 nm small unilamellar vesicles, while more complex, larger model lipid bilayers ...

Utilizing a Comprehensive Immunoprecipitation Enrichment System to Identify an Endogenous Post-translational Modification Profile for Target Proteins

It is now well-appreciated that post-translational modifications (PTMs) play an integral role in regulating a protein&#39;s structure and function, which may be essential for a given protein&#39;s role both physiologically and pathologically. Enrichment of PTMs is often necessary when investigating the PTM status of a target protein, because PTMs are often transient and relatively low in abundance. Many pitfalls are encountered when enriching for a PTM of a target protein, such as buffer incompatibility, the target protein antibody is not IP-compatible, loss of PTM signal, and others. The degree of difficulty is magnified when investigating multiple PTMs like acetylation, ubiquitination, SUMOylation 2/3, and tyrosine phosphorylation for a given target protein. Studying a combination of these PTMs may be necessary, as crosstalk between PTMs is prevalent and critical for protein regulation. Often, these PTMs are studied in different lysis buffers and with unique inhibitor compositions. To simplify the process, a unique denaturing lysis system was developed that effectively isolates and preserves these four PTMs; thus, enabling investigation of potential crosstalk in a single lysis system. A unique filter system was engineered to remove contaminating genomic DNA from the lysate, which is a problematic by-product of denaturing buffers. Robust affinity matrices targeting each of the four PTMs were developed in concert with the buffer system to maximize the enrichment and detection of the endogenous states of these four PTMs. This comprehensive PTM detection toolset streamlines the process of obtaining critical information about whether a protein is modified by one or more of these PTMs.

Investigating multiple, endogenous post-translational modifications for a target protein can be extremely challenging. Techniques described here utilize an optimized lysis buffer and filter system developed with specific PTM-targeting, affinity matrices to detect the acetylation, ubiquitination, SUMOylation 2/3, and tyrosine phosphorylation modifications for a target protein using a single, streamlined system.

It is now well-appreciated that post-translational modifications (PTMs) play an integral role in regulating a protein&#39;s structure and function, which ...

Titration ELISA as a Method to Determine the Dissociation Constant of Receptor Ligand Interaction

The dissociation constant describes the interaction between two partners in the binding equilibrium and is a measure of their affinity. It is a crucial parameter to compare different ligands, e.g., competitive inhibitors, protein isoforms and mutants, for their binding strength to a binding partner. Dissociation constants are determined by plotting concentrations of bound versus free ligand as binding curves. In contrast, titration curves, in which a signal that is proportional to the concentration of bound ligand is plotted against the total concentration of added ligand, are much easier to record. The signal can be detected spectroscopically and by enzyme-linked immunosorbent assay (ELISA). This is exemplified in a protocol for a titration ELISA that measures the binding of the snake venom-derived rhodocetin to its immobilized target domain of &#945;2&#946;1 integrin. Titration ELISAs are versatile and widely used. Any pair of interacting proteins can be used as immobilized receptor and soluble ligand, provided that both proteins are pure, and their concentrations are known. The difficulty so far has been to determine the dissociation constant from a titration curve. In this study, a mathematical function underlying titration curves is introduced. Without any error-prone graphical estimation of a saturation yield, this algorithm allows processing of the raw data (signal intensities at different concentrations of added ligand) directly by mathematical evaluation via non-linear regression. Thus, several titration curves can be recorded simultaneously and transformed into a set of characteristic parameters, among them the dissociation constant and the concentration of binding-active receptor, and they can be evaluated statistically. When combined with this algorithm, titration ELISAs gain the advantage of directly presenting the dissociation constant. Therefore, they may be used more efficiently in the future.

A detailed protocol to perform a titration ELISA is described. Moreover, a novel algorithm is presented to evaluate titration ELISAs and to obtain a dissociation constant of binding of a soluble ligand to a microtiter plate-immobilized receptor.

The dissociation constant describes the interaction between two partners in the binding equilibrium and is a measure of their affinity. It is a crucial ...

A Semi-Quantitative Drug Affinity Responsive Target Stability (DARTS) assay for studying Rapamycin/mTOR interaction

Drug Affinity Responsive Target Stability (DARTS) is a robust method for detection of novel small molecule protein targets. It can be used to verify known small molecule-protein interactions and to find potential protein targets for natural products. Compared with other methods, DARTS uses native, unmodified, small molecules and is simple and easy to operate. In this study, we further enhanced the data analysis capabilities of the DARTS experiment by monitoring the changes in protein stability and estimating the affinity of protein-ligand interactions. The protein-ligand interactions can be plotted into two curves: a proteolytic curve and a dose-dependence curve. We have used the mTOR-rapamycin interaction as an exemplary case for establishment of our protocol. From the proteolytic curve we saw that the proteolysis of mTOR by pronase was inhibited by the presence of rapamycin. The dose-dependency curve allowed us to estimate the binding affinity of rapamycin and mTOR. This method is likely to be a powerful and simple method for accurately identifying novel target proteins and for the optimization of drug target engagement.

A Semi-Quantitative Drug Affinity Responsive Target Stability DARTS assay for studying Rapamycin/mTOR interaction

In this study, we enhanced the data analysis capabilities of the DARTS experiment by monitoring the changes in protein stability and estimating the affinity of protein-ligand interactions. The interactions can be plotted into two curves: a proteolytic curve and a dose-dependence curve. We have used mTOR-rapamycin interaction as an exemplary case.

Drug Affinity Responsive Target Stability (DARTS) is a robust method for detection of novel small molecule protein targets. It can be used to verify known ...

A Customizable Approach for the Enzymatic Production and Purification of Diterpenoid Natural Products

Diterpenoids form a diverse class of small molecule natural products that are widely distributed across the kingdoms of life and have critical biological functions in developmental processes, interorganismal interactions, and environmental adaptation. Due to these various bioactivities, many diterpenoids are also of economic importance as pharmaceuticals, food additives, biofuels, and other bioproducts. Advanced genomics and biochemical approaches have enabled a rapid increase in the knowledge of diterpenoid-metabolic genes, enzymes, and pathways. However, the structural complexity of diterpenoids and the narrow taxonomic distribution of individual compounds in often only a single species remain constraining factors for their efficient production. Availability of a broader range of metabolic enzymes now provide resources for producing diterpenoids in sufficient titers and purity to facilitate a deeper investigation of this important metabolite group. Drawing on established tools for microbial and plant-based enzyme co-expression, we present an easily operated and customizable protocol for the enzymatic production of diterpenoids in either Escherichia coli or Nicotiana benthamiana, and the purification of desired products via silica chromatography and semi-preparative HPLC. Using the group of maize (Zea mays) dolabralexin diterpenoids as an example, we highlight how modular combinations of diterpene synthase (diTPS) and cytochrome P450 monooxygenase (P450) enzymes can be used to generate different diterpenoid scaffolds. Purified compounds can be used in various downstream applications, such as metabolite structural analyses, enzyme structure-function studies, and in vitro and in planta bioactivity experiments.

Here we present easy to use protocols for producing and purifying diterpenoid metabolites through the combinatorial expression of biosynthetic enzymes in Escherichia coli or Nicotiana benthamiana, followed by chromatographic product purification. The resulting metabolites are suitable for various studies including molecular structure characterization, enzyme functional studies, and bioactivity assays.

Diterpenoids form a diverse class of small molecule natural products that are widely distributed across the kingdoms of life and have critical biological ...