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

Summary

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).

Abstract

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.

Introduction

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.

Protocol

1. P Domain Cloning

1. 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 vector^6,7.
   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.
2. Digest 2 µg of the transfer vector with each 1 µl BamHI (20,000 U/ml) and NotI (10,000 U/ml) restriction enzymes for 1 hr at 37 °C with manufacturer supplied buffers.
3. 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.
4. Prepare the pMalc2x expression vector by digesting 2 µg of this vector with each 1 µl BamHI (20,000 U/ml) and NotI (10,000 U/ml) restriction enzymes for 1 hr at 37 °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 °C.
5. Ligate the purified insert into the digested pMalc2x vector at the BamHI and NotI restriction sites with 1 µl 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 µl.
6. Transform 2 µl of the ligation mix into 50 µl chemically competent E. coli DH5α bacterial cells using a standard transformation protocol (10 min on ice, heat shock 45 sec at 42 °C) and grow in 600 µl S.O.C. medium for 1 hr at 37 °C. Centrifuge the transformed cells for 3 min at 1,000 x g, discard the supernatant, and resuspend the pellet in 30 µl of S.O.C. medium.
   1. Plate the transformation mix on LB-Agar plates, containing 100 µg/ml ampicillin for selection, and grow overnight at 37 °C. Select at least five colonies.
7. For each of the five colonies, inoculate 2-3 ml culture of LB-medium supplemented with 50 µg/ml ampicillin (LB-amp) and grow by shaking overnight at 160 rpm at 37 °C.
8. 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'-TCAGACTGTCGATGAAGC-3') and reverse primer (5'-GATGTGCTGCAAGGCGAT-3').

2. P Domain Expression

1. Transform 1 μl (150 ng/μl - 400 ng/μl) of the pMalc2x vector coding for the MBP-His-P domain fusion protein into 50 μl of competent E. coli BL21 cells using a standard transformation protocol (10 min on ice, heat shock 45 sec at 42 °C) and grow in 600 μl S.O.C. medium for 1 hr at 37 °C. Subculture into 120 ml of LB-amp overnight at 160 rpm and 37 °C.
2. 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 °C until the OD₆₀₀ reaches 0.4 - 0.6. Subsequently, lower the temperature to 22 °C for ~ 1 hr and then induce the protein expression with 0.66 mM of isopropyl-β-D-thiogalactopyranoside (IPTG)⁸. Grow the cells overnight at 22 °C (~ 18 hr).
   Note: The temperature can be varied, but we recommend to use 22 °C or lower.
3. Harvest the cells by centrifugation (10,543 x g, 15 min, 4 °C). Discard the supernatant and freeze the cell pellet at -20 °C.

3. 1^st Purification Step and Protease Cleavage

1. 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 μm.
   Note: For a detailed buffer preparation scheme, refer to Table 1.
2. Thaw the cell pellet from the nine liter culture and dissolve in 150 ml PBS at 4 °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.
3. Centrifuge the sonicated cell suspension (43,667 x g, 30 min, 4 °C) to separate cell debris from the supernatant containing expressed protein. Collect the supernatant and discard the pellet.
4. 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 °C with slow rotation.
5. 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).
6. Elute the MBP-His-P domain fusion protein using 250 mM imidazole buffer (Figure 3A). During elution, check the OD_280nm to verify the elution of the fusion protein (the rise in OD_280nm). Continue elution until the OD_280nm 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).
7. 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.
8. 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 °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/µl, 1 U is sufficient to cleave 100 µg 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).

4. 2^nd Purification Step

1. Equilibrate the Ni-beads from step 3.6 in 10 mM imidazole buffer.
2. 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 °C with slow rotation.
3. 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 OD_280nm reaches ~ 0.1.
   Note: The MBP-His should remain bound to the Ni-beads (Figure 3B).
4. 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 °C against GFB for subsequent SEC purification.

5. 3^rd Purification Step

1. Wash pumps and pipes of the HPLC purification system and pre-equilibrate the SEC-column (see Materials List) with GFB.
2. 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.
3. 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.

6. Crystallization of the P Domain

1. Use the P domain at ~ 3 mg/ml and 8 mg/ml for initial crystallization screening. Prepare at least 100 µl of P domain per concentration for initial screens with 384 commercially available screening conditions. Perform screening at 18 °C in a 96-well plate format, where the reservoir contains 100 µl of mother solution and a drop is composed of 0.2 µl mother solution and 0.2 µl protein.
2. 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 µl (1 µl protein + 1 µl mother solution) and 500 µl of mother solution as a reservoir volume.
3. Use optimized crystal conditions for co-crystallizing the P domain with ligands. Prepare plates as described in 6.2. Instead of 2 µl drop size, set up drops containing 1 µl mother solution, 1 µl protein, and 1 µl of ligand at a concentration of 1 mg/ml.
4. 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 model^6,10-13.
   Note: Presence of a ligand shows up as un-modeled blob of electron density.

Results

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

Discussion

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...

Materials

Name	Company	Catalog Number	Comments
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 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