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).
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.
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.
1. P Domain Cloning
2. P Domain Expression
3. 1st Purification Step and Protease Cleavage
4. 2nd Purification Step
5. 3rd Purification Step
6. Crystallization of the P Domain
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 shows representative SDS-PAGE results of MBP-His-P domain fusion protein and cleaved P domain with the corresponding schematics of the first two steps of purification. The third purification step is depicted in Figure 4 and involves a purification scheme, the elution chromatogram of purified P domain and a representative SDS-PAGE result of collected fractions. The purest fractions (according to SDS-PAGE) are pooled, concentrated, and used for X-ray crystallography.
Figure 1. Schematic of Norovirus P Domain Expression and Purification. The protocol for norovirus P domain expression and purification contains six major parts, covering cloning and expression (1 and 2), purification (3 to 5), and crystallization (6.). Red triangles represent the P domain (gene and protein), whereas blue rectangles represent the MBP-His. Ni-NTA agarose beads that are used during IMAC (3 and 4) are illustrated as big cyan spheres. The SEC beads are depicted as grey spheres.
Figure 2. Design and Cloning of the Expression Construct (EC). The P domain EC, the expression vector map, and the multiple cloning site (MCS) are shown. A) Alignment of the norovirus capsid protein (VP1) and the P domain EC illustrating the design of the EC with the C-terminal deletion (green). B) Schematic representation of the pMalc2x expression vector used for P domain expression in E. coli with ampicillin-resistance cassette (AmpR), lac-operon (lacI), mannose-binding protein (malE, MBP) and a MCS. C) Sequence of the MCS of the pMalc2x expression vector. Highlighted are the restriction enzyme cleavage sites (red boxes) and the recognition sequence LEVLFQGP for the HRV 3C protease (precision, blue box).
Figure 3. Schematic and Representative Results for the 1st and 2nd Purification Step. Purification overview and representative SDS-PAGE results are shown. A) Purification of the MBP-His-P domain fusion protein using Ni-NTA agarose beads (big cyan spheres). The 12% SDS-PAGE gel shows the MBP-His-P domain fusion protein. B) Separation of MBP-His (blue rectangle) from cleaved P domain (red triangle). Elution of cleaved P domain is analyzed by SDS-PAGE on a 12% gel.
Figure 4. Schematic and Representative Results for the 3rd Purification Step. Chromatogram of P domain elution using SEC and corresponding SDS-PAGE result. Black numbers indicate the fractions that are collected. A) Schematic of the separation on the SEC column (SEC beads are depicted as grey spheres). The 12% SDS-PAGE shows the protein present in the fractions that are collected during SEC elution and which are subsequently pooled and used for X-ray crystallography. B) The SEC chromatogram shows the measured absorbance (black line) over elution volume. The red line indicates when the P domain was injected into the SEC-column. C) Zoom in to second peak of B. Please click here to view a larger version of this figure.
Buffer name | 1 M Tris pH 7.6 | 5 M NaCl | 3.5 M Imidazole, pH 8 |
250 mM Imidazole buffer | 20 ml | 40 ml | 71 ml |
50 mM Imidazole buffer | 20 ml | 40 ml | 14 ml |
20 mM Imidazole buffer | 20 ml | 40 ml | 5.7 ml |
10 mM Imidazole buffer | 20 ml | 40 ml | 2.8 ml |
GFB (Gel filtration buffer) | 25 ml | 60 ml | — |
Table 1. Pipetting Scheme for Common Buffers used During Purification. Stock solutions of Tris-HCl, sodium chloride (NaCl), and imidazole are prepared as indicated in the header of the table. The amount of stock solution needed to prepare 1 L of the desired buffer in water is represented.
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 of norovirus carbohydrate binding. The present protocol can be adapted to a variety of proteins. However, successful implementation of this protocol depends on several factors within each part of the purification method.
Design of the expression construct is the first step of importance. We perform codon-optimization of the P domain expression construct to improve the expression yield in E. coli and remove relevant restriction sites, present within the coding region. Moreover, we remove a flexible region at the C-terminus that could be disadvantageous for protein folding during expression and protein packing during crystallization. Expression as MBP fusion protein is a mean to keep the protein in solution during expression.
Regarding soluble protein expression there are additional parameters to be considered. The E. coli BL21 strain is optimized for high yield protein expression and therefore used in this protocol. Expression is performed overnight at 22 °C and a reduced amount of IPTG is used for induction. This is favorable for the kinetics of protein expression and as a result, less protein will aggregate into inclusion bodies due to misfolding. Therefore, it is important to cool down the culture to 22 °C before induction of protein expression with IPTG. If the protein yield is not satisfactory, it is possible to further decrease the temperature and adjust the amount of IPTG.
Certain care should be taken regarding the purification columns. In principle, Ni-beads can be reused several times. However, binding capacity will be reduced over time. If the Ni-solution loses its standard blue color, the beads can be stripped and recharged using the instructions in the manufacturer's handbook. Furthermore, it is important to maintain the SEC column in proper condition and clean it regularly to allow high performance. Depending on the protein size that has to be purified a different prep grade (pg) of the SEC column should be chosen. The P domain dimer is ~ 65 kDa in size and can be well separated from impurities of ~ 100 kDa using a 75 pg column, whereas larger proteins can be better separated using a 200 pg column.
As a combination of optimized sequence design, expression, and purification procedure it is possible to gain very pure and high quality P domain using our method. Owing to the high quality of the purified P domain, additional studies are suitable, including immunization for antibody production, NMR experiments, and ELISA-based studies. Moreover, the purified P domain can be used for complex formation with Fab antibodies and Nanobodies14,15. To our knowledge, this is the first protocol that allows P domain crystallization in a high throughput manner and, using this protocol, we have determined over 20 complex structures of various norovirus and lagovirus P domains in complex with HBGAs6,16.
According to our experience, the protocol might be limited to proteins up to 65 kDa. However, capsid proteins of different caliciviruses17 and non-structural proteins, such as viral protein genome-linked (VPg), protease, and RNA dependent RNA polymerase (RdRp) were successfully expressed and purified using this method (unpublished). When applying this method to capsid proteins of other viruses, it might be necessary to vary and optimize several parameters (e.g., the imidazole concentration of the elution buffer) to gain sufficient amount of protein. In addition, different storage buffers other than GFB (e.g., PBS or TBS) can be tested for optimal protein stability.
The majority of the analyzed constructs yielded in cubic/plate-like crystals, which diffracted to high-resolutions. Therefore, the present protocol provides a tool to obtain pure protein that crystallizes well. As long as there is now robust cell culture model available, this methodology constitutes a significant step to contribute to the understanding of norovirus-host cell interaction.
The authors have nothing to disclose.
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.
P domain DNA | Life Technologies | GeneArt Gene Synthesis | |
pMalc2x vector | On request | ||
BamHI | New England Biolabs | R0136L | |
NotI | New England Biolabs | R0189L | |
T4 DNA Ligase | New England Biolabs | M0202S | |
QIAquick Gel Extraction Kit | Qiagen | 28704 | |
QIAprep Spin Miniprep Kit | Qiagen | 27104 | |
S.O.C. Medium | Life Technologies | 15544-034 | |
Econo-Column Chromatography Column | Bio-Rad | 7372512 | 2.5 cm x 10 cm, possible to use other size |
Ni-NTA Agarose | Qiagen | 30210 | |
Vivaspin 20 | GE Healthcare | various | cutoff of 10 kDa, 30 kDa and 50 kDa used |
Subcloning Efficiency DH5α Competent Cells | Life Technologies | 18265-017 | |
One Shot BL21(DE3) Chemically Competent E. coli | Life Technologies | C6000-03 | |
HRV 3C Protease | Merck Millipore | 71493 | |
HiLoad 26/600 Superdex 75 PG | GE Healthcare | 28-9893-34 | SEC column |
JCSG Core suites | Qiagen | various | 4 screens with each 96 wells |
Carbohydrates | Dextra Laboratories, UK | various | Blood group products |