33 Leyi Wang (ed.), Animal Coronaviruses, Springer Protocols Handbooks, DOI 10.1007/978-1-4939-3414-0_4, © Springer Science+Business Media New York 2016 Chapter 4 Recombinant Turkey Coronavirus Nucleocapsid Protein Expressed in Escherichia coli Chien Chang Loa , Ching Ching Wu , and Tsang Long Lin Abstract Expression and purifi cation of turkey coronavirus (TCoV) nucleocapsid (N) protein from a prokaryotic expression system as histidine-tagged fusion protein are presented in this chapter. Expression of histidine- tagged fusion N protein with a molecular mass of 57 kDa is induced with isopropyl β- D-1- thiogalactopyranoside (IPTG). The expressed N protein inclusion body is extracted and purifi ed by chromatography on nickel-agarose column to near homogeneity. The protein recovery can be 10 mg from 100 ml of bacterial culture. The purifi ed N protein is a superior source of TCoV antigen for antibody- capture ELISA for detection of antibodies to TCoV. Key words Coronavirus , Nucleocapsid protein , Recombinant protein , Cloning , Protein expression 1 Introduction Turkey coronavirus (TCoV) is the cause of an acute and highly contagious enteric disease affecting turkeys of all ages. The disease is severe in 1- to 4-week-old turkey poults [ 1]. Turkey fl ocks that recover from natural or experimental coronaviral enteritis may develop lifelong immunity [ 2]. TCoV has been recognized as an important pathogen of young turkeys. TCoV infection causes sig- nifi cant economic losses in the turkey industry due to poor feed conversion and uneven growth. Outbreaks of TCoV enteritis in turkey poults remain as a threat to the turkey industry. In order to rapidly diagnose and effectively control turkey coronaviral enteritis , development of an antibody-capture enzyme- linked immunosorbent assay ( ELISA ) for detecting antibodies to TCoV is essential. Development of ELISA for detection of TCoV infection requires large amounts of TCoV antigen . Molecular clon- ing and expression of TCoV N protein were carried out for prepa- ration of large quantities of highly purifi ed viral proteins. Coronavirus is an enveloped and positive-stranded RNA virus that possesses three major structural proteins including a 34 predominant phosphorylated nucleocapsid (N) protein, peplo- meric glycoprotein, and spike (S) protein that makes up the large surface projections of the virion, and membrane protein (M) [ 3, 4]. The N protein is abundantly produced in coronavirus-infected cells and is highly immunogenic. The N protein binds to the viral genomic RNA and composes the structural feature of helical nucleocapsid. The N protein is a preferred choice for developing a group- specifi c serologic assay because of highly conserved sequence and antigenicity. The nucleocapsid protein s of various RNA viruses, such as mumps, rabies, vesicular stomatitis, measles, Newcastle dis- ease, and infectious bronchitis (IBV) viruses, have been used as the coating antigens in diagnostic ELISA [ 5– 10]. Prokaryotic expres- sion is an economic and convenient system to prepare large amount of pure recombinant protein. In addition, the antigenic integrity of N protein expressed in prokaryotic system is expected to be main- tained due to the lack of glycosylation. This chapter describes expression and purifi cation of TCoV N protein with a prokaryotic system for preparation of a large quantity of highly purifi ed viral protein, which can be used as coating antigen for antibody-capture ELISA for serologic diagnosis of TCoV infection [ 11, 12]. 2 Materials 1. Virus source ( see Note 1). 2. RNApure reagent (GenHunter, Nashville, TN, USA). 3. Chloroform. 4. Isopropanol. 5. 70 % Ethanol. 6. Diethyl-pyrocarbonate (DEPC)-treated sterile double- distilled water (DEPC-H 2O). 7. SuperScript III fi rst-strand synthesis system for RT-PCR kit (Life Technologies/Invitrogen, Carlsbad, CA, USA). 8. RNaseOUT, a recombinant RNase inhibitor (Life Technologies/Invitrogen). 9. RNase H enzyme (Life Technologies/Invitrogen). 10. Random hexamers (Life Technologies/Invitrogen). 11. N gene primers ( see Note 2): NF, 5′-TCTTTTGCCATGGC AAGC- 3′; NR, 5′-TTGGGTACCTAAAAGTTCATTCTC-3′. 12. Taq polymerase (Promega Corp, Madison, WI, USA). 13. Pfu polymerase (Stratagene, La Jolla, CA, USA). 14. Deoxynucleotide triphosphates (dNTP) (Promega). 15. Sterile distilled water. 2.1 Construction of TCoV N Gene in the Expression Vector pTriEx Chien Chang Loa et al. 35 16. pTriEx 1.1 cloning kit (Novagen, Madison, WI, USA; EMD Millipore Corp., Billerica, MA, USA). 17. Vector primer for clone screening: TriExUP primer (Novagen; EMD Millipore Corp.). 18. QIAquick PCR product purifi cation kit (Qiagen, Valencia, CA, USA). 19. Restriction enzymes: Nco I and Kpn I (New England Biolabs, Inc., Ipswich, MA, USA) ( see Note 3). 20. Shrimp alkaline phosphatase (SAP) (Promega Corp). 21. Zymoclean gel DNA recovery kit (Zymo Research, Irvine, CA, USA). 22. Escherichia coli ( E. coli) NovaBlue competent cell (Novagen; EMD Millipore Corp.). 23. Growth medium: LB medium containing 50 μg/ml carbenicil- lin (Life Technologies, Grand Island, NY, USA). 24. QIAquick mini-prep kit (Qiagen). 25. E. coli Tuner (DE3) pLacI competent cell (Novagen; EMD Millipore Corp.). 1. Expression host E. coli cell, strain Tuner (DE3) pLacI transfor- mant with recombinant plasmid pTri-N containing N protein gene (prepared from Sects. 2.1 and 3.1). 2. Expression medium: LB medium containing 50 μg/ml car- benicillin, 34 μg/ml chloramphenicol, and 1 % glucose (Life Technologies) ( see Note 4). 3. Isopropyl β- D-1-thiogalactopyranoside (IPTG), 100 mM (Life Technologies). 4. Incubator shaker (New Brunswick Scientifi c Co., Inc., Edison, NJ, USA; Eppendorf North America, Hauppauge, NY, USA). 1. BugBuster (Novagen; EMD Millipore Corp.). 2. Benzonase (Novagen; EMD Millipore Corp.). 3. His-Bind column, pre-packed column with 1.25 ml Ni 2+ charged His-Bind resin (Novagen; EMD Millipore Corp.) ( see Note 5). 4. Binding buffer, 5 mM imidazole, 0.5 M NaCl, 20 mM Tris– HCl (pH 7.9) (Novagen; EMD Millipore Corp.) containing 6 M urea ( see Note 6). 5. Wash buffer, 20 mM imidazole, 0.5 M NaCl, 20 mM Tris– HCl (pH 7.9) (Novagen; EMD Millipore Corp.) containing 6 M urea. 6. Elute buffer, 1 M imidazole, 0.5 M NaCl, 20 mM Tris–HCl (pH 7.9) (Novagen; EMD Millipore Corp.) containing 6 M urea. 2.2 Expression of Recombinant TCoV Protein 2.3 Purifi cation of Recombinant TCoV N Protein Expression of TCoV N protein 36 7. Protein assay reagent (Bio-Rad, Hercules, CA, USA). 8. SDS-polyacrylamide gel electrophoresis and Western blotting apparatuses (Mini-Protean ® electrophoresis chamber and wet/ tank blotting system, Bio-Rad). 3 Methods 1. Total RNA was extracted from TCoV virus source by RNApure reagent. Two hundred microliters of virus solution are mixed with 1 ml of RNApure reagent and incubated on ice for 10 min ( see Note 7). 2. Add 180 μl of chloroform, mix the mixture, and vortex vigor- ously for 10 s ( see Note 8). 3. Centrifuge at 13,000 × g for 10 min at 4 °C. Carefully take the upper aqueous phase into a clean microcentrifuge tube and mix with equal volume of cold isopropanol by vortexing vigor- ously for 30 s. Incubate on ice for 10 min. 4. Centrifuge at 13,000 × g for 10 min at 4 °C. Carefully discard the supernatant without disturbing the RNA pellet. 5. Wash RNA pellet with 1 ml of cold 70 % ethanol. Incubate on ice for 5 min. 6. Centrifuge at 13,000 × g for 2 min at 4 °C. Remove ethanol. Spin briefl y and remove the residual liquid with pipette ( see Note 9). 7. Dissolve RNA pellet in 50 μl of DEPC-H 2O and a portion of it is quantifi ed by spectrophotometry (GeneQuant Pro Spectrophotometer, Amersham Pharmacia Biotech, Inc., Piscataway, NJ, USA; GeneQuant 1300 Spectrophotometer, GE Healthcare Bio-Sciences, Piscataway, NJ, USA) at 260 nm wavelength ( see Note 10). 8. Mix 8 μl (1 pg to 5 μg) of RNA with 1 μl (50 ng/μl) random hexamer and 1 μl (10 mM) dNTP in a total volume of 10 μl. 9. Incubate at 65 °C for 5 min and sit on ice for 1 min. 10. Add 10 μl of SuperScript III cDNA Synthesis Mix (containing 2 μl 10× RT buffer, 4 μl (25 mM) MgCl 2, 2 μl (0.1 M) DTT, 1 μl (40 U/μl) RNaseOUT, 1 μl (200 U/μl) SuperScript III RT enzyme) to each RNA/primer mixture. 11. Incubate at 25 °C for 10 min followed by 50 °C for 50 min. 12. Terminate the reverse transcription (RT) reaction at 85 °C for 5 min and chill on ice ( see Note 11). 13. Add 2 μl of the above RT mixture to the PCR amplifi cation reaction (100 μl) with primers NF and NR. A mix of Taq and Pfu at 10:1 is recommended to maintain PCR fi delity (Table 1). 3.1 Construction of TCoV N Gene in the Expression Vector pTriEx Chien Chang Loa et al. 37 14. PCR cyclic parameters: 94 °C for 10 s for denaturation, 58 °C for 30 s for annealing, 72 °C for 2 min for extension for 35 cycles followed by 72 °C for 10-min fi nal extension. 15. PCR product is purifi ed by QIAquick PCR purifi cation kit and recovered in 16 μl of sterile distilled water (pH 7.0–8.5). 16. Add all 16 μl of purifi ed PCR product to the restriction enzyme (RE) digestion with 1 μl (10 units/μl) Nco I, 1 μl (10 units/ μl) Kpn I, and 2 μl 10× buffer in one reaction of 20 μl. Incubate at 37 °C for 4 h. 17. Set up the same RE digestion for 1 μg of vector pTriEx 1.1 vector plasmid. 18. For dephosphorylation of digested vector, add 1 μl (1 unit) of SAP to the 20 μl of digestion reaction mixture. Incubate at 37 °C for 30 min. 19. Gel purify both digestion reactions for PCR product and plasmid pTriEx by Zymoclean gel DNA recovery kit. Recover each digested DNA prep in 10 μl of sterile distilled water ( see Note 12). 20. Set up two ligation reactions (pTriEx 1.1 cloning kit) with 1 μl of pTriEx vector and 1 or 4 μl of PCR product (insert) DNA in a reaction of 10 μl as demonstrated in Table 2. Incubate at 16 °C for 15 min ( see Note 13). 21. Take 1 μl of ligation reaction for transformation to NovaBlue competent cell. Plate 20 and 100 μl of the transformation mix on growth medium plates ( see Note 14). 22. Colony screening by PCR with primers TriExUp and NR. The PCR cycling parameters are the same as above for N gene Table 1 Preparation of reaction mixture for turkey coronavirus nucleocapsid protein gene polymerase chain reaction Components Volumes (μl) 10× Buffer 10 MgCl 2 (25 mM/ml) 6 dNTP (10 mM each) 2 Primer NF (100 ng/μl) 4 Primer NR (100 ng/μl) 4 RT reaction mixture 20 DEPC-H 2O 53 Taq/Pfu enzyme 1 Expression of TCoV N protein 38 amplifi cation (step 14). The PCR product of correct clone is about 1500 bp. 23. The selected clone is grown in growth medium. Plasmids are purifi ed by QIAquick mini-prep kit and sequenced to confi rm that the inserted TCoV N gene is in frame with the vector- defi ned open reading frame at both its N- and C-termini. 24. The recombinant plasmid containing the entire TCoV N pro- tein gene (pTri-N) is transformed to competent E. coli strain Tuner (DE3) pLacI. Transformants are grown in expression medium. 1. A starter culture of expression host bacteria cells is prepared in 3 ml of expression medium. Cells are incubated at 37 °C with shaking at 250 rpm in an incubator shaker overnight, to an OD of 600 nm approximately 0.5 ( see Note 15). 2. The entire 3 ml culture is added to 100 ml of fresh expression medium and incubated at 37 °C with shaking at 250 rpm in an incubator shaker until the OD reached 0.5–1.0 at 600 nm wavelength ( see Note 16). 3. The cultures are induced by addition of IPTG to a fi nal con- centration of 1 mM (add 1 ml of sterile 100 mM IPTG solu- tion). The induced cultures are incubated at 37 °C with shaking at 250 rpm in an incubator shaker for another 4 h. 4. The bacterial cultures are harvested by centrifugation at 10,000 × g for 10 min at 4 °C. The cell pellet can be immedi- ately processed or frozen stored at −20 or −80 °C until pro- cessed ( see Note 17). 1. Resuspend the cell pellet completely in 5 ml of BugBuster reagent per gram of wet cell weight ( see Note 18). 2. The nuclease reagent Benzonase is added with 1 μl (25 units) for every ml of BugBuster reagent used. 3.2 Expression of Recombinant TCoV N Protein 3.3 Purifi cation of Recombinant TCoV N Protein Table 2 Preparation of ligation reaction mixture for recombinant plasmid carrying turkey coronavirus nucleocapsid protein gene Components Volumes (μl) Vector x Insert y Sterile water z Ligation premix (2×) 5 ( x + y + z = 5 μl) Chien Chang Loa et al. 39 3. Incubate at room temperature with slow rotation for 20 min. 4. Centrifuge at 16,000 × g for 20 min at 4 °C. The soluble super- natant is discarded and the insoluble fraction (containing inclusion bodies) is dissolved in 10 ml of binding buffer ( see Note 19). 5. Equilibrate His-Bind column with 10 ml of binding buffer. Allow the entire buffer volume to fl ow through. 6. Apply the protein solution (dissolved inclusion bodies) to the column. 7. Wash the column with 10 ml of binding buffer. 8. Wash the column with 10 ml of wash buffer. 9. Elute the recombinant N protein from the column with 5 ml of elute buffer ( see Note 20). 10. The concentration of purifi ed N protein is determined by pro- tein assay reagent. The yield of purifi ed N protein is about 10 mg from a 100 ml of culture ( see Note 21). 11. The purifi ed N protein is further confi rmed by SDS- polyacrylamide gel electrophoresis and Western blotting as a single protein band with a molecular mass of about 57 kDa. 4 Notes 1. Homogenates of infected intestines can be the virus source. For better results, further purifi cation of intestinal homoge- nates containing TCoV with 40–60 % sucrose gradient is rec- ommended. TCoV/IN/94 (GenBank accession number EU022525) has been used as the stock virus for expression of recombinant TCoV N protein in our laboratory. 2. Primers NF and NR containing restriction sites Nco I and Kpn I, respectively. The amplifi ed product containing the entire open reading frame of TCoV N protein gene (1230 bp). 3. Nco I and Kpn I are typical restriction enzymes. The diges- tions by both enzymes can be conducted in a single reaction with compatible buffer. For example, the two enzymes from New England Biolabs (Ipswich, MA) can be performed in NE Buffer 1.1. 4. Ampicillin antibiotic marker is on the expression vector pTriEx and chloramphenicol antibiotic marker is on plasmid pLacI in the expression host strain Tuner cells. Carbenicillin is recom- mended to be in place of ampicillin for better stability for pH changes throughout the bacterial cultures. 5. The binding capacity of 1.25 ml of His-Bind resin is 10 mg of target protein per column. As for any affi nity chromatography, Expression of TCoV N protein 40 the best purity of target protein is achieved when the amount of protein extract is near the binding capacity. 6. The purpose of 6 M urea is to improve resolution of the sticky inclusion bodies. The presence of 6 M urea does not affect binding of His-Bind resin to target N protein. 7. The suggested ratio of RNApure reagent to sample is 10:1. Excess amount of RNApure reagent has no negative impact. The lower ratio (5:1) in this step is intended to obtain higher concentration of viral RNA in the fi nal supernatants. If the upper aqueous phase after centrifugation at step 3 is more than half of the total volume, there is not enough RNApure reagent added. The appropriate reagent amount may be adjusted. Chloroform is applied at 150 μl for every milliliter of lysate. 8. The sample mixture with chloroform at this step can be stored at −70 °C or even lower temperature before proceeding to the next step. 9. Optional: Inverting the tube for 5–10 min for air-drying of RNA pellet is a helpful tip to completely remove any residual ethanol that may interfere the following RT reaction. 10. It is critical to make sure that the jellylike RNA pellet is com- pletely dissolved into solution by repeat pipetting. The volume (50 μl) of DEPC-H 2O may be adjusted according to RNA pel- let size to achieve appropriate concentrations. Concentration can be estimated by taking 1 μl of the RNA solution into 1 ml of water. Read at 260 nm. 1 OD260 = 40 μg. The RNA qual- ity can be further examined by OD 260/280 and 260/230 ratio. The ratio of 260/280 about 2.0 is considered as pure for RNA, while 1.8 is considered pure for DNA . The expected 260/230 ratio is around 2.0–2.2 for pure nucleic acid. If the ratio is appreciably lower, it may indicate the presence of pro- tein, phenol, or other contaminants with strong absorption near 280 or 230 nm. RNA should be stored at −70 °C or even lower temperature. 11. The synthesized cDNA in the RT reaction can be stored at −20 °C or even lower temperature until used. 12. PCR product may be purifi ed. The vector must be gel purifi ed due to the long digested fragment size above 30 bp. 13. The molar ratio between vector and insert is suggested at 1:2 to 1:5. The volumes in this step are illustrated for initial exploration. The concentration of digested vector and insert can be estimated by OD 260 or agarose gel electrophoresis with known amount of DNA of similar size in adjacent wells. The ligation reaction mixture can be stored at 4 °C until used for transformation or at −20 °C for longer term. Chien Chang Loa et al. 41 14. After plating, the leftover transformation mix can be stored at 4 °C for further plating in the following days at different amount if needed. 15. The starter culture can be prepared from a fresh colony on a plate or directly from a glycerol storage stock. An OD around 0.5 represents a culture at log phase when the cells are at the best condition to expand and for protein expression. 16. This usually takes about 2–3 h to reach the OD range. The higher the OD of starter culture in the previous step, the shorter the time to reach this OD range. 17. The centrifuge tubes should be weighed before and after col- lection of cell pellet for estimation of wet pellet amount and the volume of BugBuster to be applied in the next step. Frozen storage of cell pellets may improve the extraction effi ciency of BugBuster reagents through the freeze/thaw cycle. 18. It is important to completely resuspend the cell pellets for the best results of BugBuster extraction. Higher volume of BugBuster reagent does not have adverse effect. Roughly 10–20 ml of BugBuster reagent should be enough for cell pel- lets collected from a 100 ml of culture. BugBuster reagent can be added directly to frozen cell pellets. There is no need to wait for the temperature to return to room temperature. Protease inhibitors may be added at this step but usually not necessary. 19. It is critical but somewhat diffi cult to completely dissolve the sticky inclusion bodies. Repeat pipetting up and down until the protein solution is homogeneous. Any undissolved parti- cles will clot the His-Bind column and affect the purifi cation process. It is advisable to centrifuge the dissolved inclusion body protein solution at 5000–10,000 × g for 10–15 min at 4 °C for clarifi cation before application to the column. 20. Eluate may be collected in fractions such as 0.5 or 1 ml each fraction. 21. The presence of 6 M urea is compatible with the protein assay reagent. The assay range can be adjusted for protein concen- trations from low μg/ml to 1 mg/ml with different assay for- mat. The protein concentration of the target N protein eluate as obtained following this process is about 1–2 mg/ml. The presence of 6 M urea has no adverse effect on plate coating for ELISA performance. Given the coating concentration of N protein at 20 μg/ml, the eluate is usually diluted in coating buffer for at least 1:10 to reduce the urea content to less than 600 mM and, subsequently, further diminish any possible effect on ELISA performance. Accordingly, the purifi ed N protein eluate can be directly applied to the ELISA assay for detection of antibodies to TCoV. Expression of TCoV N protein 42 Acknowledgements The protocol “Recombinant turkey coronavirus nucleocapsid pro- tein expressed in Escherichia coli” detailed in this chapter had been successfully carried out in the authors’ studies on characterization and immunology of turkey coronaviral enteritis . Those studies were in part fi nancially supported by USDA, North Carolina Poultry Federation, and/or Indiana Department of Agriculture and technically assisted by Drs. Tom Brien and David Hermes, Mr. Tom Hooper, and Ms. Donna Schrader for clinical and diagnostic investigation, virus isolation and propagation, and animal experimentation. References 1. Nagaraja KV, Pomeroy BS (1997) Coronaviral enteritis of turkeys (blue comb disease). In: Calnek BW, Barnes HJ, Beard CW et al (eds) Diseases of poultry, 10th edn. Iowa state University Press, Ames, IA 2. 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