The funding for this study was provided by the CHS foundation. We acknowledge the protein crystallization platform within the excellence cluster CellNetworks of the University of Heidelberg for crystal screening and the European Synchrotron Radiation Facility for provision of synchrotron radiation facilities.

1. P Domain Cloning
<ol>
	<li>Determine the P domain coding region by sequence alignment of norovirus strains (e.g., GII.10 strain, GenBank: AF504671, pdb-ID: 3ONU)6. Moreover, remove the flexible region at the C-terminal end of the P domain (Figure 2A). Codon-optimize the DNA for E. coli expression and include BamHI (N-terminal) and NotI (C-terminal) restriction sites in order to sub-clone the P domain coding region into the pMalc2x expression vector6,7. 
	Note: The P domain coding region is optimized and synthesized by a commercial service. The P domain coding region (insert) is approximately 1 kb in length and delivered in a standard transfer vector.</li>
	<li>Digest 2 &#181;g of the transfer vector with each 1 &#181;l BamHI (20,000 U/ml) and NotI (10,000 U/ml) restriction enzymes for 1 hr at 37 &#176;C with manufacturer supplied buffers.</li>
	<li>Separate the digested insert on a 1% agarose gel for 20 min at 135 V and purify the insert DNA from the gel using a commercial kit.</li>
	<li>Prepare the pMalc2x expression vector by digesting 2 &#181;g of this vector with each 1 &#181;l BamHI (20,000 U/ml) and NotI (10,000 U/ml) restriction enzymes for 1 hr at 37 &#176;C. Purify the vector from an agarose gel as described above (1.3). Note: Both samples (1.2 and 1.4) can be stored at -20 &#176;C.</li>
	<li>Ligate the purified insert into the digested pMalc2x vector at the BamHI and NotI restriction sites with 1 &#181;l T4-DNA ligase (400,000 U/ml) for 15 min at room temperature (RT) (Figure 2B and 2C). Use at least 20 ng of the pMalc2x vector and a vector:insert ratio 1:3 (molecular weight). The ligation mix is usually ~ 20 &#181;l.</li>
	<li>Transform 2 &#181;l of the ligation mix into 50 &#181;l chemically competent E. coli DH5&#945; bacterial cells using a standard transformation protocol (10 min on ice, heat shock 45 sec at 42 &#176;C) and grow in 600 &#181;l S.O.C. medium for 1 hr at 37 &#176;C. Centrifuge the transformed cells for 3 min at 1,000 x g, discard the supernatant, and resuspend the pellet in 30 &#181;l of S.O.C. medium.
	<ol>
		<li>Plate the transformation mix on LB-Agar plates, containing 100 &#181;g/ml ampicillin for selection, and grow overnight at 37 &#176;C. Select at least five colonies.</li>
	</ol>
	</li>
	<li>For each of the five colonies, inoculate 2-3 ml culture of LB-medium supplemented with 50 &#181;g/ml ampicillin (LB-amp) and grow by shaking overnight at 160 rpm at 37 &#176;C.</li>
	<li>Extract the plasmids from the overnight culture using a commercial kit. Verify the presence of the P domain insert by sequencing with a pMalc2x forward primer (5&#39;-TCAGACTGTCGATGAAGC-3&#39;) and reverse primer (5&#39;-GATGTGCTGCAAGGCGAT-3&#39;).</li>
</ol>
2. P Domain Expression
<ol>
	<li>Transform 1 &#956;l (150 ng/&#956;l - 400 ng/&#956;l) of the pMalc2x vector coding for the MBP-His-P domain fusion protein into 50 &#956;l of competent E. coli BL21 cells using a standard transformation protocol (10 min on ice, heat shock 45 sec at 42 &#176;C) and grow in 600 &#956;l S.O.C. medium for 1&#160;hr at 37 &#176;C. Subculture into 120 ml of LB-amp overnight at 160 rpm and 37 &#176;C.</li>
	<li>Inoculate nine liters (e.g., 6 x 5 L flasks with 1.5 L medium each) of LB-amp with the subculture (1:100). Grow the cells shaking at 160 rpm and 37 &#176;C until the OD600 reaches 0.4 - 0.6. Subsequently, lower the temperature to 22 &#176;C for ~ 1 hr and then induce the protein expression with 0.66 mM of isopropyl-&#946;-D-thiogalactopyranoside (IPTG)8. Grow the cells overnight at 22 &#176;C (~ 18 hr). 
	Note: The temperature can be varied, but we recommend to use 22 &#176;C or lower.</li>
	<li>Harvest the cells by centrifugation (10,543 x g, 15 min, 4 &#176;C). Discard the supernatant and freeze the cell pellet at -20 &#176;C.</li>
</ol>
3. 1st Purification Step and Protease Cleavage
<ol>
	<li>Prepare buffers that are used during the protein purification steps from stock solutions to guarantee reproducibility and stability of the experiments. Prepare four different buffers for the immobilized metal ion affinity chromatography (IMAC), each containing a different concentration of imidazole (10 mM, 20 mM, 50 mM, and 250 mM). For the SEC, prepare a gel-filtration buffer (GFB) with a higher salt concentration, but without imidazole. Use deionized water and filter all buffers before use with a pore size of 0.45 &#956;m. 
	Note: For a detailed buffer preparation scheme, refer to Table 1.</li>
	<li>Thaw the cell pellet from the nine liter culture and dissolve in 150 ml PBS at 4 &#176;C. Sonicate the cell suspension three times for 2 min (power 130 W, amplitude 20%, pulse frequency 50%) to disrupt the cells. Keep the cell suspension on ice during sonication.</li>
	<li>Centrifuge the sonicated cell suspension (43,667 x g, 30 min, 4 &#176;C) to separate cell debris from the supernatant containing expressed protein. Collect the supernatant and discard the pellet.</li>
	<li>Wash and equilibrate 10 ml (=1 column volume [CV]) slurry of Nickel (Ni)-NTA agarose beads with 10 mM imidazole buffer in a chromatography column. Add the equilibrated Ni beads to the supernatant from step 3.3 containing expressed MBP-His-P domain fusion protein and incubate for 30 min at 4 &#176;C with slow rotation.</li>
	<li>After incubation, apply the entire Ni-bead-protein mixture to a chromatography column. Wash the column slowly with each 5 CVs of 10 mM, 20 mM, and 50 mM imidazole buffers, starting with 10 mM, then 20 mM and last 50 mM (Figure 3A).</li>
	<li>Elute the MBP-His-P domain fusion protein using 250 mM imidazole buffer (Figure 3A). During elution, check the OD280nm to verify the elution of the fusion protein (the rise in OD280nm). Continue elution until the OD280nm drops to ~ 0.1. Wash the beads with excessive amounts of 250 mM imidazole buffer (at least 10 CVs), followed by at least 10 CVs of 10 mM imidazole buffer. Save the beads for the second purification step (section 4).</li>
	<li>Verify the presence of the MBP-His-P domain fusion protein with SDS-PAGE using a 12% SDS-polyacrylamide gel (10 x 8 cm)9 (Figure 3A). Perform gel electrophoresis at 45 A and 200 V for 45 min.</li>
	<li>Concentrate (e.g., using a commercial concentrator) the eluted MBP-His-P domain fusion protein to a final concentration of ~ 3 mg/ml. Cleave the MBP-His-P domain fusion with HRV-3C protease during dialysis against 2 L of 10 mM imidazole buffer (~ 1:100) overnight at 4 &#176;C (Figure 3A). Depending on the final volume of concentrated protein, perform dialysis in a dialysis cassette or dialysis tubing. 
	Note: The amount of HRV-3C protease for protein cleavage is calculated according to the specific protease activity (2 U/&#181;l, 1 U is sufficient to cleave 100 &#181;g of protein) and amount of eluted fusion protein that varies on the expression level and can be estimated from the SDS-PAGE result (3.7).</li>
</ol>
4. 2nd Purification Step
<ol>
	<li>Equilibrate the Ni-beads from step 3.6 in 10 mM imidazole buffer.</li>
	<li>Incubate the dialyzed protein from step 3.7 (containing cleaved P domain, MBP protein, and HRV-protease) with the equilibrated Ni-beads (4.1) for 30 min at 4 &#176;C with slow rotation.</li>
	<li>Apply the Ni-bead mixture to a column and collect the flow-through (cleaved P domain) (Figure 3B). Measure the concentration of protein as it comes off the column until the OD280nm reaches ~ 0.1. 
	Note: The MBP-His should remain bound to the Ni-beads (Figure 3B).</li>
	<li>Check the presence of cleaved P domain using SDS-PAGE with a 12% gel as described above (Figure 3B). Concentrate the eluted P domain to ~ 3 mg/ml and dialyze overnight at 4 &#176;C against GFB for subsequent SEC purification.</li>
</ol>
5. 3rd Purification Step
<ol>
	<li>Wash pumps and pipes of the HPLC purification system and pre-equilibrate the SEC-column (see Materials List) with GFB.</li>
	<li>Inject the P domain to the column at a flow-rate of 1 ml/min using a superloop (up to 12 ml) or loop (up to 3 ml), depending on the volume of the concentrated sample. After the injection is finished, increase the flow rate to 2.5 ml/min.</li>
	<li>As the OD increases and the P domain comes off the column, collect fractions of 1.5 ml. Check fractions using SDS-PAGE with a 12% gel (Figure 4A and 4B). Pool only the purest fractions and concentrate to ~ 3 mg/ml and ~ 8 mg/ml. 
	Note: After ~110 ml (void volume) most impurities are eluted from a SEC column with 320 ml bed volume. The P domain is usually eluted as a dimer. Elution time/volume of the P domain dimer is dependent on the prep grade (pg) of the SEC column.</li>
</ol>
6. Crystallization of the P Domain
<ol>
	<li>Use the P domain at ~ 3 mg/ml and 8 mg/ml for initial crystallization screening. Prepare at least 100 &#181;l of P domain per concentration for initial screens with 384 commercially available screening conditions. Perform screening at 18 &#176;C in a 96-well plate format, where the reservoir contains 100 &#181;l of mother solution and a drop is composed of 0.2 &#181;l mother solution and 0.2 &#181;l protein.</li>
	<li>Repeat and optimize successful crystallization conditions. Therefore, use 15-well plates that contain 3 rows. Set up the first row with 100% mother solution, the second row with 90% mother solution and 10% water, and the third row with 80% mother solution and 20% water. Use a drop size of 2 &#181;l (1 &#181;l protein + 1 &#181;l mother solution) and 500 &#181;l of mother solution as a reservoir volume.</li>
	<li>Use optimized crystal conditions for co-crystallizing the P domain with ligands. Prepare plates as described in 6.2. Instead of 2 &#181;l drop size, set up drops containing 1 &#181;l mother solution, 1 &#181;l protein, and 1 &#181;l of ligand at a concentration of 1 mg/ml.</li>
	<li>Collect data sets of single crystals using synchrotron radiation. Perform molecular replacement using published P domain structures with a high sequence similarity as initial search model6,10-13. 
	Note: Presence of a ligand shows up as un-modeled blob of electron density.</li>
</ol>

<ol>
	<li>Ahmed, S. 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=Global+prevalence+of+norovirus+in+cases+of+gastroenteritis:+a+systematic+review+and+meta-analysis.">Global prevalence of norovirus in cases of gastroenteritis: a systematic review and meta-analysis.</a> Lancet Infect. Dis. 14, 725-730 (2014).</li><li>Prasad, B. V., Matson, D. O., Smith, A. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three-dimensional+structure+of+calicivirus.">Three-dimensional structure of calicivirus.</a> J. Mol. Biol. 240, 256-264 (1994).</li><li>Choi, J. M., Hutson, A. M., Estes, M. K., Prasad, B. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Atomic+resolution+structural+characterization+of+recognition+of+histo-blood+group+antigens+by+Norwalk+virus.">Atomic resolution structural characterization of recognition of histo-blood group antigens by Norwalk virus.</a> Proc. Natl. Acad. Sci. U.S.A. 105, 9175-9180 (2008).</li><li>Marionneau, 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=Norwalk+virus+binds+to+histo-blood+group+antigens+present+on+gastroduodenal+epithelial+cells+of+secretor+individuals.">Norwalk virus binds to histo-blood group antigens present on gastroduodenal epithelial cells of secretor individuals.</a> Gastroenterology. 122, 1967-1977 (2002).</li><li>Jones, M. 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=Enteric+bacteria+promote+human+and+mouse+norovirus+infection+of+B+cells.">Enteric bacteria promote human and mouse norovirus infection of B cells.</a> Science. 346, 755-759 (2014).</li><li>Hansman, G. 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=Crystal+structures+of+GII.10+and+GII.12+norovirus+protruding+domains+in+complex+with+histo-blood+group+antigens+reveal+details+for+a+potential+site+of+vulnerability.">Crystal structures of GII.10 and GII.12 norovirus protruding domains in complex with histo-blood group antigens reveal details for a potential site of vulnerability.</a> J. Virol. 85, 6687-6701 (2011).</li><li>Fath, 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=Multiparameter+RNA+and+codon+optimization:+a+standardized+tool+to+assess+and+enhance+autologous+mammalian+gene+expression.">Multiparameter RNA and codon optimization: a standardized tool to assess and enhance autologous mammalian gene expression.</a> PLoS One. 6 (e17596), (2011).</li><li>Jacob, F., Monod, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+regulatory+mechanisms+in+the+synthesis+of+proteins.">Genetic regulatory mechanisms in the synthesis of proteins.</a> J. Mol. Biol. 3, 318-356 (1961).</li><li>Laemmli, U. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cleavage+of+structural+proteins+during+the+assembly+of+the+head+of+bacteriophage+T4.">Cleavage of structural proteins during the assembly of the head of bacteriophage T4.</a> Nature. 227, 680-685 (1970).</li><li>Kabsch, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Automatic+Processing+of+Rotation+Diffraction+Data+from+Crystals+of+Initially+Unknown+Symmetry+and+Cell+Constants.">Automatic Processing of Rotation Diffraction Data from Crystals of Initially Unknown Symmetry and Cell Constants.</a> J. Appl. Crystallogr. 26, 795-800 (1993).</li><li>Mccoy, A. 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=Phaser+crystallographic+software.">Phaser crystallographic software.</a> J. Appl. Crystallogr. 40, 658-674 (2007).</li><li>Emsley, P., Lohkamp, B., Scott, W. G., Cowtan, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Features+and+development+of+Coot.">Features and development of Coot.</a> Acta Crystallogr. Sect. D-Biol. Crystallogr. 66, 486-501 (2010).</li><li>Adams, P. D., 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=PHENIX:+a+comprehensive+Python-based+system+for+macromolecular+structure+solution.">PHENIX: a comprehensive Python-based system for macromolecular structure solution.</a> Acta Crystallogr. Sect. D-Biol. Crystallogr. 66, 213-221 (2010).</li><li>Koromyslova, A. D., Hansman, G. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nanobody+binding+to+a+conserved+epitope+promotes+norovirus+particle+disassembly.">Nanobody binding to a conserved epitope promotes norovirus particle disassembly.</a> J. Virol. 89, 2718-2730 (2015).</li><li>Hansman, G. 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=Structural+basis+for+broad+detection+of+genogroup+II+noroviruses+by+a+monoclonal+antibody+that+binds+to+a+site+occluded+in+the+viral+particle.">Structural basis for broad detection of genogroup II noroviruses by a monoclonal antibody that binds to a site occluded in the viral particle.</a> J. Virol. 86, 3635-3646 (2012).</li><li>Singh, B. K., Leuthold, M. M., Hansman, G. 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The authors have nothing to disclose.

Here, we describe a protocol for the expression and purification of norovirus P domains in high quality and quantity. Noroviruses are not well studied and structural data are continuously needed. To our knowledge, P domain production using other protocols (e.g., GST-tagged P domains) has been problematic, so far, and sufficient structural data on norovirus-host interaction have been missing. With the method described here, we have recently contributed significantly to the understanding of the molecular details o...

Human noroviruses are the major cause of acute gastroenteritis worldwide1. These viruses belong to the Caliciviridae family, of which there are at least five genera, including Norovirus, Sapovirus, Lagovirus, Vesivirus, and Nebovirus. Despite their high impact on the healthcare system and wide distribution, the study of human noroviruses is hampered by the lack of a robust cell culture system. To date, there are no approved vaccines or antiviral strategies available.
The norovirus major capsid protein, termed VP1, can be divided into a shell (S) domain and a protruding (P) domain2. The P domain is connected to the S domain by a flexible hinge (H) region. The S domain forms a scaffold around the viral RNA, whereas the P domain forms the outmost part of the viral capsid. The P domain assembles into biologically relevant dimers when expressed in bacteria. The P dimer interacts with carbohydrate structures, termed histo-blood group antigens (HBGAs) that are present as soluble antigens in saliva and found on certain host cells3. The P domain-HBGA interaction is thought to be important for infection4. Indeed, a recent report revealed the importance of synthetic HBGAs or HBGA-expressing bacteria for human norovirus infection in vitro5.
Current studies regarding the host cell attachment of noroviruses are mainly performed with virus-like particles (VLPs) that can be expressed in insect cells or with recombinant P domains expressed in Escherichia coli (E. coli). To understand the P domain-HBGA interactions at atomic resolution, P domain-HBGA complex structures can be solved using X-ray crystallography. Here, we describe a protocol for P domain expression and purification that allows production of P domain in high quantity and quality to be used for X-ray crystallography. Moreover, this method can be applied for other calicivirus P domains and non-structural proteins.
The P domain is codon-optimized for E. coli expression and cloned into a standard transfer vector. The P domain is then re-cloned into an expression vector that encodes a polyhistidine (His) tag and a mannose-binding protein (MBP) that are followed by a protease cleavage site. The MBP-His-P domain fusion protein is expressed in E. coli, followed by three purification steps. The MBP-His-P domain fusion protein is purified using immobilized metal ion affinity chromatography (IMAC). Next, the fusion protein is cleaved with human rhinovirus (HRV)-3C protease and the P domain is separated from the MBP-His by an additional IMAC purification step. Lastly, the P domain is purified using size exclusion chromatography (SEC). The purified P domain can then be used for X-ray crystallography. Screening of protein crystallization conditions is performed with commercially available screening kits using different P domain protein concentrations. Crystal growth is observed and the most promising conditions are optimized.
With the methods described here, it is possible to go from gene to protein to structure within less than four weeks. Therefore, our method of P domain expression, purification, and crystallization is suitable to study norovirus-host interaction at the molecular level and provide important data to assist in up-to-date vaccine design and drug screening.

<table>
	<tbody>
		<tr>
			<td>P domain DNA</td>
			<td>Life Technologies</td>
			<td></td>
			<td>GeneArt Gene Synthesis</td>
		</tr>
		<tr>
			<td>pMalc2x&#160; vector</td>
			<td>On request</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>BamHI</td>
			<td>New England Biolabs</td>
			<td>R0136L</td>
			<td></td>
		</tr>
		<tr>
			<td>NotI</td>
			<td>New England Biolabs</td>
			<td>R0189L</td>
			<td></td>
		</tr>
		<tr>
			<td>T4 DNA Ligase</td>
			<td>New England Biolabs</td>
			<td>M0202S</td>
			<td></td>
		</tr>
		<tr>
			<td>QIAquick Gel Extraction Kit</td>
			<td>Qiagen</td>
			<td>28704</td>
			<td></td>
		</tr>
		<tr>
			<td>QIAprep Spin Miniprep Kit</td>
			<td>Qiagen</td>
			<td>27104</td>
			<td></td>
		</tr>
		<tr>
			<td>S.O.C. Medium</td>
			<td>Life Technologies</td>
			<td>15544-034</td>
			<td></td>
		</tr>
		<tr>
			<td>Econo-Column Chromatography Column</td>
			<td>Bio-Rad</td>
			<td>7372512</td>
			<td>2.5 cm x 10 cm, possible to use other size</td>
		</tr>
		<tr>
			<td>Ni-NTA Agarose</td>
			<td>Qiagen</td>
			<td>30210</td>
			<td></td>
		</tr>
		<tr>
			<td>Vivaspin 20</td>
			<td>GE Healthcare</td>
			<td>various</td>
			<td>cutoff of 10 kDa, 30 kDa and 50 kDa used</td>
		</tr>
		<tr>
			<td>Subcloning Efficiency DH5&#945; Competent Cells</td>
			<td>Life Technologies</td>
			<td>18265-017</td>
			<td></td>
		</tr>
		<tr>
			<td>One Shot BL21(DE3) Chemically Competent E. coli</td>
			<td>Life Technologies</td>
			<td>C6000-03</td>
			<td></td>
		</tr>
		<tr>
			<td>HRV 3C Protease</td>
			<td>Merck Millipore</td>
			<td>71493</td>
			<td></td>
		</tr>
		<tr>
			<td>HiLoad 26/600 Superdex 75 PG</td>
			<td>GE Healthcare</td>
			<td>28-9893-34</td>
			<td>SEC column</td>
		</tr>
		<tr>
			<td>JCSG Core suites</td>
			<td>Qiagen</td>
			<td>various</td>
			<td>4 screens with each 96 wells</td>
		</tr>
		<tr>
			<td>Carbohydrates</td>
			<td>Dextra Laboratories, UK</td>
			<td>various</td>
			<td>Blood group products</td>
		</tr>
	</tbody>
</table>

production of human norovirus protruding domains in e. coli for x-ray crystallography

The norovirus capsid is composed of a single major structural protein, termed VP1. VP1 is subdivided into a shell (S) domain and a protruding (P) domain. The S domain forms a contiguous scaffold around the viral RNA, whereas the P domain forms viral spikes on the S domain and contains determinants for antigenicity and host-cell interactions. The P domain binds carbohydrate structures, i.e., histo-blood group antigens, which are thought to be important for norovirus infections. In this protocol, we describe a method for producing high quality norovirus P domains in high yields. These proteins can then be used for X-ray crystallography and ELISA in order to study antigenicity and host-cell interactions.
The P domain is firstly cloned into an expression vector and then expressed in bacteria. The protein is purified using three steps that involve immobilized metal-ion affinity chromatography and size exclusion chromatography. In principle, it is possible to clone, express, purify, and crystallize proteins in less than four weeks, which makes this protocol a rapid system for analyzing newly emerging norovirus strains.

The schematic of the described protocol is depicted in Figure 1. The protocol covers 6 major parts that include cloning of the target gene, expression, a three-step purification, and crystallization. Figure 2 illustrates the design of the expression construct (EC) and characteristics of the pMalc2x expression vector. The sequence of the multiple cloning site (MCS) of the pMalc2x vector shows restriction and protease cleavage sites. Figure 3</stron...

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Production of Human Norovirus Protruding Domains in E. coli for X-ray Crystallography

Here, we describe a method to express and purify high quality norovirus protruding (P) domains in E. coli for use in X-ray crystallography studies. This method can be applied to other calicivirus P domains, as well as non-structural proteins, i.e., viral protein genome-linked (VPg), protease, and RNA dependent RNA polymerase (RdRp).

Production of Human Norovirus Protruding Domains in E. coli for X-ray Crystallography

The norovirus capsid is composed of a single major structural protein, termed VP1. VP1 is subdivided into a shell (S) domain and a protruding (P) domain. ...

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8.6K Views.  University of Heidelberg and the German Cancer Research Center. Here, we describe a method to express and purify high quality norovirus protruding (P) domains in E. coli for use in X-ray crystallography studies. This method can be applied to other calicivirus P domains, as well as non-structural proteins, i.e., viral protein genome-linked (VPg), protease, and RNA dependent RNA polymerase (RdRp).

Video: Production of Human Norovirus Protruding Domains in E. coli for X-ray Crystallography

Transformation of Plasmid DNA into E. coli Using the Heat Shock Method

Transformation of plasmid DNA into E. coli using the heat shock method is a basic technique of molecular biology. It consists of inserting a foreign plasmid or ligation product into bacteria. This video protocol describes the traditional method of transformation using commercially available chemically competent bacteria from Genlantis. After a short incubation in ice, a mixture of chemically competent bacteria and DNA is placed at 42&deg;C for 45 seconds (heat shock) and then placed back in ice. SOC media is added and the transformed cells are incubated at 37&deg;C for 30 min with agitation. To be assured of isolating colonies irrespective of transformation efficiency, two quantities of transformed bacteria are plated. This traditional protocol can be used successfully to transform most commercially available competent bacteria. The turbocells from Genlantis can also be used in a novel 3-minute transformation protocol, described in the instruction manual.

Transformation of plasmid DNA into E. coli using the heat shock method is a basic technique of molecular biology. It consists of inserting a foreign ...

Non-invasive 3D-Visualization with Sub-micron Resolution Using Synchrotron-X-ray-tomography

Little is known about the internal organization of many micro-arthropods with body sizes below 1 mm. The reasons for that are the small size and the hard cuticle which makes it difficult to use protocols of classical histology. In addition, histological sectioning destroys the sample and can therefore not be used for unique material. Hence, a non-destructive method is desirable which allows to view inside small samples without the need of sectioning.
We used synchrotron X-ray tomography at the European Synchrotron Radiation Facility (ESRF) in Grenoble (France) to non-invasively produce 3D tomographic datasets with a pixel-resolution of 0.7&micro;m. Using volume rendering software, this allows us to reconstruct the internal organization in its natural state without the artefacts produced by histological sectioning. These date can be used for quantitative morphology, landmarks, or for the visualization of animated movies to understand the structure of hidden body parts and to follow complete organ systems or tissues through the samples.

We used synchrotron X-ray tomography at the European Synchrotron Radiation Facility (ESRF) to non-invasively produce 3D tomographic datasets with a pixel-resolution of 0.7&micro;m. Using volume rendering software, this allows the reconstruction of internal structures in their natural state without the artefacts produced by histological sectioning.

Little is known about the internal organization of many micro-arthropods with body sizes below 1 mm. The reasons for that are the small size and the hard ...

Visualization of UV-induced Replication Intermediates in E. coli using Two-dimensional Agarose-gel Analysis

Inaccurate replication in the presence of DNA damage is responsible for the majority of cellular rearrangements and mutagenesis observed in all cell types and is widely believed to be directly associated with the development of cancer in humans. DNA damage, such as that induced by UV irradiation, severely impairs the ability of replication to duplicate the genomic template accurately. A number of gene products have been identified that are required when replication encounters DNA lesions in the template. However, a remaining challenge has been to determine how these proteins process lesions during replication in vivo. Using Escherichia coli as a model system, we describe a procedure in which two-dimensional agarose-gel analysis can be used to identify the structural intermediates that arise on replicating plasmids in vivo following UV-induced DNA damage. This procedure has been used to demonstrate that replication forks blocked by UV-induced damage undergo a transient reversal that is stabilized by RecA and several gene products associated with the RecF pathway. The technique demonstrates that these replication intermediates are maintained until a time that correlates with the removal of the lesions by nucleotide excision repair and replication resumes.

Visualization of UV-induced Replication Intermediates in E. coli using Two-dimensional Agarose-gel Analysis

We present a procedure by which two-dimensional agarose-gel analysis can be used to identify the structure of replication intermediates that occur following UV irradiation.

Inaccurate replication in the presence of DNA damage is responsible for the majority of cellular rearrangements and mutagenesis observed in all cell types ...

Protein Crystallization for X-ray Crystallography

Using the three-dimensional structure of biological macromolecules to infer how they function is one of the most important fields of modern biology. The availability of atomic resolution structures provides a deep and unique understanding of protein function, and helps to unravel the inner workings of the living cell. To date, 86% of the Protein Data Bank (rcsb-PDB) entries are macromolecular structures that were determined using X-ray crystallography.
To obtain crystals suitable for crystallographic studies, the macromolecule (e.g. protein, nucleic acid, protein-protein complex or protein-nucleic acid complex) must be purified to homogeneity, or as close as possible to homogeneity. The homogeneity of the preparation is a key factor in obtaining crystals that diffract to high resolution (Bergfors, 1999; McPherson, 1999).
Crystallization requires bringing the macromolecule to supersaturation. The sample should therefore be concentrated to the highest possible concentration without causing aggregation or precipitation of the macromolecule (usually 2-50 mg/ mL). Introducing the sample to precipitating agent can promote the nucleation of protein crystals in the solution, which can result in large three-dimensional crystals growing from the solution. There are two main techniques to obtain crystals: vapor diffusion and batch crystallization. In vapor diffusion, a drop containing a mixture of precipitant and protein solutions is sealed in a chamber with pure precipitant. Water vapor then diffuses out of the drop until the osmolarity of the drop and the precipitant are equal (Figure 1A). The dehydration of the drop causes a slow concentration of both protein and precipitant until equilibrium is achieved, ideally in the crystal nucleation zone of the phase diagram. The batch method relies on bringing the protein directly into the nucleation zone by mixing protein with the appropriate amount of precipitant (Figure 1B). This method is usually performed under a paraffin/mineral oil mixture to prevent the diffusion of water out of the drop.
Here we will demonstrate two kinds of experimental setup for vapor diffusion, hanging drop and sitting drop, in addition to batch crystallization under oil.

The 3-D structure of a molecule provides a unique understanding of how the molecule functions. The principal method for structure determination at near-atomic resolution is X-ray crystallography. Here, we demonstrate the current methods for obtaining three-dimensional crystals of any given macromolecule that are suitable for structure determination by X-ray crystallography.

Using the three-dimensional structure of biological macromolecules to infer how they function is one of the most important fields of modern biology. The ...

Mutagenesis and Functional Selection Protocols for Directed Evolution of Proteins in E. coli

The efficient generation of genetic diversity represents an invaluable molecular tool that can be used to label DNA synthesis, to create unique molecular signatures, or to evolve proteins in the laboratory. Here, we present a protocol that allows the generation of large (>1011) mutant libraries for a given target sequence. This method is based on replication of a ColE1 plasmid encoding the desired sequence by a low-fidelity variant of DNA polymerase I (LF-Pol I). The target plasmid is transformed into a mutator strain of E. coli and plated on solid media, yielding between 0.2 and 1 mutations/kb, depending on the location of the target gene. Higher mutation frequencies are achieved by iterating this process of mutagenesis. Compared to alternative methods of mutagenesis, our protocol stands out for its simplicity, as no cloning or PCR are involved. Thus, our method is ideal for mutational labeling of plasmids or other Pol I templates or to explore large sections of sequence space for the evolution of activities not present in the original target. The tight spatial control that PCR or randomized oligonucleotide-based methods offer can also be achieved through subsequent cloning of specific sections of the library. Here we provide protocols showing how to create a random mutant library and how to establish drug-based selections in E. coli to identify mutants exhibiting new biochemical activities.

Mutagenesis and Functional Selection Protocols for Directed Evolution of Proteins in E. coli

Here we demonstrate a simple protocol to create a random mutant library for a given target sequence. We show how this method, which is performed in vivo in Escherichia coli, can be coupled with functional selections to evolve new enzymatic activities.

The efficient generation of genetic diversity represents an invaluable molecular tool that can be used to label DNA synthesis, to create unique molecular ...

A Convenient and General Expression Platform for the Production of Secreted Proteins from Human Cells

Recombinant protein expression in bacteria, typically E. coli, has been the most successful strategy for milligram quantity expression of proteins. However, prokaryotic hosts are often not as appropriate for expression of human, viral or eukaryotic proteins due to toxicity of the foreign macromolecule, differences in the protein folding machinery, or due to the lack of particular co- or post-translational modifications in bacteria. Expression systems based on yeast (P. pastoris or S. cerevisiae) 1,2, baculovirus-infected insect (S. frugiperda or T. ni) cells 3, and cell-free in vitro translation systems 2,4 have been successfully used to produce mammalian proteins. Intuitively, the best match is to use a mammalian host to ensure the production of recombinant proteins that contain the proper post-translational modifications. A number of mammalian cell lines (Human Embryonic Kidney (HEK) 293, CV-1 cells in Origin carrying the SV40 larget T-antigen (COS), Chinese Hamster Ovary (CHO), and others) have been successfully utilized to overexpress milligram quantities of a number of human proteins 5-9. However, the advantages of using mammalian cells are often countered by higher costs, requirement of specialized laboratory equipment, lower protein yields, and lengthy times to develop stable expression cell lines. Increasing yield and producing proteins faster, while keeping costs low, are major factors for many academic and commercial laboratories.
Here, we describe a time- and cost-efficient, two-part procedure for the expression of secreted human proteins from adherent HEK 293T cells. This system is capable of producing microgram to milligram quantities of functional protein for structural, biophysical and biochemical studies. The first part, multiple constructs of the gene of interest are produced in parallel and transiently transfected into adherent HEK 293T cells in small scale. The detection and analysis of recombinant protein secreted into the cell culture medium is performed by western blot analysis using commercially available antibodies directed against a vector-encoded protein purification tag. Subsequently, suitable constructs for large-scale protein production are transiently transfected using polyethyleneimine (PEI) in 10-layer cell factories. Proteins secreted into litre-volumes of conditioned medium are concentrated into manageable amounts using tangential flow filtration, followed by purification by anti-HA affinity chromatography. The utility of this platform is proven by its ability to express milligram quantities of cytokines, cytokine receptors, cell surface receptors, intrinsic restriction factors, and viral glycoproteins. This method was also successfully used in the structural determination of the trimeric ebolavirus glycoprotein 5,10.
In conclusion, this platform offers ease of use, speed and scalability while maximizing protein quality and functionality. Moreover, no additional equipment, other than a standard humidified CO2 incubator, is required. This procedure may be rapidly expanded to systems of greater complexity, such as co-expression of protein complexes, antigens and antibodies, production of virus-like particles for vaccines, or production of adenoviruses or lentiviruses for transduction of difficult cell lines.

In the post-human genomics era, the availability of recombinant proteins in native conformations is crucial to structural, functional and therapeutic research and development. Here, we describe a test- and large-scale protein expression system in human embryonic kidney 293T cells that can be used to produce a variety of recombinant proteins.

Recombinant protein expression in bacteria, typically E. coli, has been the most successful strategy for milligram quantity expression of proteins. ...

3D Printing of Preclinical X-ray Computed Tomographic Data Sets

Three-dimensional printing allows for the production of highly detailed objects through a process known as additive manufacturing. Traditional, mold-injection methods to create models or parts have several limitations, the most important of which is a difficulty in making highly complex products in a timely, cost-effective manner.1 However, gradual improvements in three-dimensional printing technology have resulted in both high-end and economy instruments that are now available for the facile production of customized models.2 These printers have the ability to extrude high-resolution objects with enough detail to accurately represent in vivo images generated from a preclinical X-ray CT scanner. With proper data collection, surface rendering, and stereolithographic editing, it is now possible and inexpensive to rapidly produce detailed skeletal and soft tissue structures from X-ray CT data. Even in the early stages of development, the anatomical models produced by three-dimensional printing appeal to both educators and researchers who can utilize the technology to improve visualization proficiency. 3, 4 The real benefits of this method result from the tangible experience a researcher can have with data that cannot be adequately conveyed through a computer screen. The translation of pre-clinical 3D data to a physical object that is an exact copy of the test subject is a powerful tool for visualization and communication, especially for relating imaging research to students, or those in other fields. Here, we provide a detailed method for printing plastic models of bone and organ structures derived from X-ray CT scans utilizing an Albira X-ray CT system in conjunction with PMOD, ImageJ, Meshlab, Netfabb, and ReplicatorG software packages.

Using modern plastic extrusion and printing technologies, it is now possible to quickly and inexpensively produce physical models of X-ray CT data taken in a laboratory. The three -dimensional printing of tomographic data is a powerful visualization, research, and educational tool that may now be accessed by the preclinical imaging community.

Three-dimensional printing allows for the production of highly detailed objects through a process known as additive manufacturing.  Traditional, ...

Efficient Production and Purification of Recombinant Murine Kindlin-3 from Insect Cells for Biophysical Studies

Kindlins are essential coactivators, with talin, of the cell surface receptors&#160;integrins and also participate in integrin outside-in signalling, and the control of gene transcription in the cell nucleus. The kindlins are ~75 kDa multidomain proteins and bind to an NPxY motif and upstream T/S cluster of the integrin &#946;-subunit cytoplasmic tail. The hematopoietically-important kindlin isoform, kindlin-3, is critical for platelet aggregation during thrombus formation, leukocyte rolling in response to infection and inflammation and osteoclast podocyte formation in bone resorption. Kindlin-3&#39;s role in these processes has resulted in extensive cellular and physiological studies. However, there is a need for an efficient method of acquiring high quality milligram quantities of the protein for further studies. We have developed a protocol, here described, for the efficient expression and purification of recombinant murine kindlin-3 by use of a baculovirus-driven expression system in Sf9 cells yielding sufficient amounts of high purity full-length protein to allow its biophysical characterization. The same approach could be taken in the study of the other mammalian kindlin isoforms.

Kindlins are fundamental to cell adhesion through integrins but studies of them have been hampered by the difficulty encountered in expressing them recombinantly in bacterial hosts. We describe here methods for their efficient production in baculovirus-infected insect cells.

Kindlins are essential coactivators, with talin, of the cell surface receptors&#160;integrins and also participate in integrin outside-in signalling, and ...

Green Fluorescent Protein-based Expression Screening of Membrane Proteins in Escherichia coli

The production of recombinant membrane proteins for structural and functional studies remains technically challenging due to low levels of expression and the inherent instability of many membrane proteins once solubilized in detergents. A protocol is described that combines ligation independent cloning of membrane proteins as GFP fusions with expression in Escherichia coli detected by GFP fluorescence. This enables the construction and expression screening of multiple membrane protein/variants to identify candidates suitable for further investment of time and effort. The GFP reporter is used in a primary screen of expression by visualizing GFP fluorescence following SDS polyacrylamide gel electrophoresis (SDS-PAGE). Membrane proteins that show both a high expression level with minimum degradation as indicated by the absence of free GFP, are selected for a secondary screen. These constructs are scaled and a total membrane fraction prepared and solubilized in four different detergents. Following ultracentrifugation to remove detergent-insoluble material, lysates are analyzed by fluorescence detection size exclusion chromatography (FSEC). Monitoring the size exclusion profile by GFP fluorescence provides information about the mono-dispersity and integrity of the membrane proteins in different detergents. Protein: detergent combinations that elute with a symmetrical peak with little or no free GFP and minimum aggregation are candidates for subsequent purification. Using the above methodology, the heterologous expression in E. coli of SED (shape, elongation, division, and sporulation) proteins from 47 different species of bacteria was analyzed. These proteins typically have ten transmembrane domains and are essential for cell division. The results show that the production of the SEDs orthologues in E. coli was highly variable with respect to the expression levels and integrity of the GFP fusion proteins. The experiment identified a subset for further investigation.

Green Fluorescent Protein-based Expression Screening of Membrane Proteins in Escherichia coli

A streamlined approach to screening for the expression of recombinant membrane proteins in Escherichia coli based on fusion to green fluorescent protein is presented.

The production of recombinant membrane proteins for structural and functional studies remains technically challenging due to low levels of expression and ...

Phage-mediated Delivery of Targeted sRNA Constructs to Knock Down Gene Expression in E. coli

RNA-mediated knockdowns are widely used to control gene expression. This versatile family of techniques makes use of short RNA (sRNA) that can be synthesized with any sequence and designed to complement any gene targeted for silencing. Because sRNA constructs can be introduced to many cell types directly or using a variety of vectors, gene expression can be repressed in living cells without laborious genetic modification. The most common RNA knockdown technology, RNA interference (RNAi), makes use of the endogenous RNA-induced silencing complex (RISC) to mediate sequence recognition and cleavage of the target mRNA. Applications of this technique are therefore limited to RISC-expressing organisms, primarily eukaryotes. Recently, a new generation of RNA biotechnologists have developed alternative mechanisms for controlling gene expression through RNA, and so made possible RNA-mediated gene knockdowns in bacteria. Here we describe a method for silencing gene expression in E. coli that functionally resembles RNAi. In this system a synthetic phagemid is designed to express sRNA, which may designed to target any sequence. The expression construct is delivered to a population of E. coli cells with non-lytic M13 phage, after which it is able to stably replicate as a plasmid. Antisense recognition and silencing of the target mRNA is mediated by the Hfq protein, endogenous to E. coli. This protocol includes methods for designing the antisense sRNA, constructing the phagemid vector, packaging the phagemid into M13 bacteriophage, preparing a live cell population for infection, and performing the infection itself. The fluorescent protein mKate2 and the antibiotic resistance gene chloramphenicol acetyltransferase (CAT) are targeted to generate representative data and to quantify knockdown effectiveness.

Phage-mediated Delivery of Targeted sRNA Constructs to Knock Down Gene Expression in E. coli

We describe a method to knock down gene expression in a growing population of E. coli cells using sequence-targeted sRNA expression cassettes delivered by an M13 phagemid vector.