Indian Journal of Biochemistry & Biophysics Vol. 45, April 2008, pp 98-105 Optimized expression, solubilization and purification of nuclear inclusion protein b of Cardamom mosaic virus T Jebasingh, T Jacob, M Shah, D Das, S Krishnaswamy and R Usha* School of Biotechnology, Madurai Kamaraj University, Madurai-625021, Tamil Nadu, India Received 5 September 2007; revised 28 February 2008 All RNA viruses encode an RNA-dependent RNA polymerase (RdRP) that is required for replication of the viral genome. Nuclear inclusion b (NIb) gene codes for the RdRp in Potyviridae viruses. In this study, expression, solubilization and purification of NIb protein of Cardamom mosaic virus (CdMV) is reported. The objective of the present study was to express and purify the NIb protein of CdMV on a large scale for structural characterization, as the structure of the RdRp from a plant virus is yet to be determined. However, the expression of NIb protein with hexa-histidine tag in Escherichia coli led to insoluble aggregates. Out of all the approaches [making truncated versions to reduce the size of protein; replacing an amino acid residue likely to be involved in hydrophobic intermolecular interactions with a hydrophilic one; expressing the protein along with chaperones; expression in Origami cells for proper disulphide bond formation, in E. coli as a fusion with maltose-binding protein (MBP) and in Nicotiana tabacum] to obtain the RdRp in a soluble form, only expression in E. coli as a fusion with MBP and its expression in N. tabacum were successful. The NIb expressed in plant or as a fusion with MBP in E. coli can be scaled up for further work. Keywords: RNA-dependent RNA polymerase, Nuclear inclusion b protein, Cardamom mosaic virus, Inclusion bodies, Expression, Solubilization Cardamom mosaic virus (CdMV), the causative agent proteins, namely Qβ replicase subunit2, poliovirus 3D of the widespread ‘katte’ disease, is a member of polymerase3-5, hepatitis C virus NS5B protein3,6-7 and Macluravirus genus of Potyviridae1. Morphologically, tobacco vein mottling virus (TVMV) NIb8. The three- the disease is characterized by mosaic-like light green dimensional structure has been determined for the stripes on the leaf lamina and pseudostem. There is a RdRps of three viruses — poliovirus9, bacteriophage marked reduction in the size as well as the production Ф610 and hepatitis C11. A number of sequence motifs of the capsules. Cardamom plants infected by CdMV common among the putative RdRps of several animal show significant reductions in the yield by 70–100% in and plant positive strand RNA viruses have been one to three years. Infected plants are stunted in identified12. The RdRps carry a highly conserved growth. All the members of Potyviridae have a single- ‘GDD’ motif and are able to catalyze template and strand positive sense RNA genome of ~10 kb. The primer-dependent poly (U) formation8. nuclear inclusion b (NIb) protein of Potyviridae is In order to characterize the NIb of CdMV, we report assigned as the RNA-dependent RNA polymerase here the expression of the NIb with hexa-histidine tag (RdRp), which plays a critical role in the life cycle of in E. coli. The inclusion bodies (IBs) formed have been RNA viruses that replicate without any DNA partially solubilized with 8 M urea and purified by intermediate. passing through a Ni-NTA column. The large size, The catalytic activity of RdRp has been hydrophobic nature, a large number of cysteines and demonstrated biochemically in only a handful of viral the intermolecular interactions have been ruled out as the causes of aggregation. The soluble form of NIb has ________________ been obtained by fusion of the truncated NIb to *Corresponding author maltose-binding protein (MBP) and expressing it in E-mail: [email protected] Tel: 91-452-2458230; Fax: 91-452-2459105 Nicotiana tabacum. Abbreviations: CdMV, Cardamom mosaic virus; CP, coat protein; IBs, inclusion bodies; IPTG, isopropyl-beta-D-1-thiogalactopyrano- Materials and Methods side; MBP, maltose-binding protein; Nia/ NIb, nuclear inclusion Cloning and expression of NIb proteins a and b; RdRp, RNA-dependent RNA polymerase; TF, trigger factor; TVMV, tobacco vein mottling virus; WSMV, wheat The NIb coding region of CdMV (1.7 kb) was streak mosaic virus. amplified from pJS3 (which contained a 3.0 kb JEBASINGH et al: EXPRESSION AND PURIFICATION OF NUCLEAR INCLUSION PROTEIN B 99 fragment comprising the partial nuclear inclusion a monoclonal antibody against the histidine tag or a (NIa) protease, complete NIb and partial coat protein polyclonal antiserum raised against NIb as the (CP) coding region of CdMV (Accession number for primary antiserum. NIb: AJ345002). The PCR was performed for 30 cycles (94oC, 1 min melting, 55oC, 50 s for annealing, Truncations in NIb protein 70oC, 1.5 min for polymerization) with NIbEF1 (5’ A number of truncations were made in NIb GGGTTGCAAATGTTTGAAG 3’) and NIbER1 (Fig. 1). The construct pNIb2 was made from pNIb1 with HindIII digestion, which released a fragment (5’GGATCCTATTGTCTTGGTG CTGTTGGC 3’) th primers by using the enzyme elongase (Invitrogen). from 913 nucleotide position of NIb gene to end of The DNA (50 ng) and 20 pico moles each of the the NIb. The remaining fragment was then self-ligated above primers were used in a 100 µl reaction volume. to derive pNIb2 (Fig. 1). The construct pNIb3 was The amplicon was cloned at StuI site in pHT7 vector13 made by releasing a 1.1 kb fragment from pNIb1 with to derive pNIb1. The pNIb1 was transformed into DraI and XbaI and cloning at StuI and XbaI sites of BL21 (DE3) E. coli cells. For expression, 1% of an pHT7 (pNIb3). The construct pNIb4 was made by over-night grown culture of BL21 (DE3) cells, releasing 0.45 kb fragment from pNIb1 with XbaI and harboring pNIb1 was inoculated in LB medium and cloning at the corresponding site of pHT7 to derive o pNIb4. The truncations pNIb2 and pNIb3 had the stop the culture was grown till 0.6 O.D600 at 37 C. Protein expression was induced by the addition of isopropyl- codon from the vector and pNIb4 from the NIbER1 β-D-1-thiogalactopyranoside (IPTG) to a final primer. All these constructs were transferred to BL21 concentration of 0.2 mM and the cells were further (DE3) cells and the expression was carried out as grown for 2.5 h at 37oC. mentioned above for NIb1. Ni-NTA purification Site-directed mutagenesis The NIb expressing cell pellets were resuspended A point mutation was made in NIb by replacing a th in a sonication buffer containing 8 M urea in 10 mM Phe at 525 position with a Glu. The mutagenic β-mercaptoethanol, 0.1% SDS, 0.1 M Tris, 0.01 M primers (NIbFE.For-5’ GTTTACGAGGATCCA- NaH2PO4, pH 8.0, sonicated by four pulses for 10 s TGTCAA3’ and NIbFE.Rev-5’ CTTGACATGG- each and incubated for 30 min. The cell extract was ATCCTCGTA 3’ along with the flanking primers centrifuged at 10,000 g for 10 min and the supernatant (NIbEF1 and NIbER1) were used to make the point 14 was mixed with Ni-NTA agarose and incubated for 30 mutation in NIb by overlap extension PCR . The min. A column was washed with increasing italicized sequences corresponding to BamHI concentrations of imidazole (10, 7 and 3 column restriction site were added to the mutagenic primers volumes of 10 mM, 50 mM and 100 mM imidazole without affecting the ORF (open reading frame) to respectively) in the sonication buffer (8 M urea in confirm the mutation. The first PCR was performed o o 10 mM β-mercaptoethanol, 0.1% SDS, 0.1 M Tris, for 30 cycles (94 C, 1 min for melting, 55 C, 50 s for o 0.01 M NaH2PO4, pH 8.0) and finally the bound annealing, 70 C, 1.5 min for polymerization) with protein was eluted with 200 mM imidazole. NIbEF1 primer and mutagenic primer (NIbFE.Rev) by using elongase. The second PCR was performed o o Production of anti-NIb antiserum for 30 cycles (94 C, 1 min for melting, 55 C, 40 s for A sensitizing dose of 1 mg of purified NIb was annealing, 70oC, 1.5 min for polymerization) with given intramuscularly to a rabbit with complete mutagenic primer NIbFE. For and NIbER1 by using adjuvant, followed by two subcutaneous injections elongase enzyme. The pNIb1 (50 ng) was used as the with 500 µg of protein in incomplete adjuvant. A final template for these two PCRs. The above two PCR dose of 500 µg of NIb was given intradermally with products were purified and used as the template for incomplete adjuvant. All injections were given at third PCR, performed with the flanking primers weekly intervals. Serum was collected ten days after (NIbEF1 and NIbER1) with the same conditions as the final injection. for the full length NIb amplification. The amplicon was cloned at StuI site in pHT7 vector to derive Western blotting pNIb6. The point mutation in pNIb6 was confirmed The purified 200 mM imidazole fraction was by BamHI digestion (Fig. 7) and sequencing before subjected to Western analysis with either a using it for expression in BL21 (DE3) E. coli. 100 INDIAN J. BIOCHEM. BIOPHYS., VOL. 45, APRIL 2008 Co-expressing NIb with chaperones into the Agrobacterium strain LBA4404. Leaves of BL21 cells harboring a pair of expression Nicotiana tabacum (2-4 weeks old and axenically plasmids–pNIb1 and the plasmid pG-Tf2 (which grown in tissue culture) were used for pGA-NIbHIs 15 could express trigger factor (TF) and GroEL-GroES transformation as described17. From the transformed together under the tetracycline-inducible promoter leaf discs, plants were raised and screened for NIb (Pzt-1)) were grown in medium containing ampicillin gene.