Name: production of human norovirus protruding domains in e. coli for x-ray crystallography
Uploaded: 2016-04-19T14:00:00
Description: Watch this Scientific Journal Video about Production of Human Norovirus Protruding Domains in E. coli for X-ray Crystallography at JoVE.com

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. 
	...

<ol>
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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...

Watch this Scientific Journal Video about Production of Human Norovirus Protruding Domains in E. coli for X-ray Crystallography at JoVE.com

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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