Debarko banerji sacnas ppresentation


Published on

Cloning and expression of the Nodamura virus RNA-dependent RNA polymerase

Poster presentation at Society for the Advancement of Chicanos and Native Americans in Science (SACNAS) National Conference, October 2012, Seatltle, WA

Published in: Technology
  • Be the first to comment

  • Be the first to like this

No Downloads
Total views
On SlideShare
From Embeds
Number of Embeds
Embeds 0
No embeds

No notes for slide

Debarko banerji sacnas ppresentation

  1. 1. Cloning and expression of theNodamura virus RNA-dependent RNApolymeraseDebarko Banerji1, Alexandra Navarro1,Vincent U. Gant, Jr.1, and Kyle L.Johnson1,21Department of Biological Sciences and 2BorderBiomedical Research Center, The University of Texas atEl Paso, El Paso, TX
  2. 2. Nodamura virus (NoV) is the type species of the Alphanodavirus Genus of the Family Nodaviridae, whichalso includes Flock House virus (FHV). These are small icosahedral viruses with bipartite, positive-strandRNA genomes. Nodaviruses can replicate their genomes in a wide variety of cells, including baby hamsterkidney (BHK21) cells and cells of the yeast Saccharomyces cerevisiae, which makes them an attractivemodel system for RNA replication studies. NoV is unique among the nodaviruses in that it can infect bothinsects and mammals, resulting in hind segment/limb paralysis and death (1). Genome replication andcapsid functions are divided onto separate genome segments: RNA 1 and RNA 2, respectively. NoV RNA 1encodes Protein A (hereafter referred to as NA), a 110 kDa RNA-dependent RNA polymerase (RdRp) that isessential for infectivity. The NoV and FHV RdRps, which share only 44% sequence identity (2), show uniquetemplate specificity in that the two RdRps do not replicate one another’s RNA templates.Currently, the mechanism of initiation of NA RNA synthesis is unknown, i.e. we do not know how NArecognizes the template RNA. NA has yet to be purified to homogeneity, however preliminary data fromour lab has shown that NA can be purified using affinity chromatography. Purified NA will allow us toconduct in vitro RNA replication studies (3,4,5) as well as elucidate the 3D structure. We hypothesize thatmembrane targeted expression of NA using the SRP-SecY pathway in E. coli will yield membrane-solubleRdRp that can then be purified to homogeneity to carry out further studies (6). In this project we aredeveloping a recombinant DNA based protocol for the overexpression and purification of NA. from E. coli.INTRODUCTION
  3. 3. The purpose of this project is to develop areliable protocol for the overexpression andpurification of recombinant NA (NoV RdRp) tobe used for downstream studies of NAenzymatic activity.PURPOSE
  4. 4. Our laboratory studies the mechanism of viral RNA replication, using Nodamura virus(NoV) as a model, due to its genetic simplicity, tremendous levels of RNA amplification,and ability to replicate in a wide variety of host cells. NoV contains a bipartite positive-strand RNA genome. The larger segment (RNA1) encodes the RNA-dependent RNApolymerase (RdRp) that catalyzes replication of the viral genome, while RNA2, encodesthe viral capsid protein precursor, which is dispensable for RdRp function.We are developing an in vitro replication system to study the specific mechanism bywhich the viral RdRp recognizes its RNA template. As a first step, we will expressrecombinant RdRp with an N-terminal 6-HIS tag in E. coli from an IPTG-inducible T7promoter. We used PCR to introduce an NcoI site, a start codon, and a 6-HIS tag to the5’-end of the RdRp ORF and to introduce a 3’-terminal HindIII site after the RdRp stopcodon. The PCR products were subcloned into pGEM-T-Easy vectors and confirmed byDNA sequencing. The 5’ and 3’ terminal fragments isolated from these subclones will beligated to an internal fragment containing the majority of the RdRp ORF and introducedinto the pET45b(+) vector. The vector will be used to transform competent T7-EXPRESSE. coli cells and pilot studies will be performed to determine the optimal expressionconditions. The expressed protein will be purified by affinity chromatography andassayed for RdRp activity. The purified protein will allow us to determine the replicationinitiation mechanism used by this enzyme.ABSTRACT
  5. 5. Construction of pET45b(+)-SRP-NA expression plasmid.5 and 3 Insert Fragments. The 5 and 3 fragments of the insert sequence of DNA were PCR-amplified fromparental plasmid pTXB1-SRP-NoV1, which contains the gene for recombinant NA as follows: N-terminal E. coliSRP signal (required to target protein synthesis to the membrane) followed by a Factor X cleavage site. Inamplifying the 5 fragment, primers were designed so as to introduce on the 5 end of the recombinant gene, aBbsI restriction site which upon digestion would leave an NcoI overhang (BbsI is a remote cutter) to allowligation into the destination vector, followed by an AUG start codon, followed by the sequence for a 6Xhistidine tag (to enable purification via Ni2+-NTA affinity chromatography) (3). In amplifying the 3 fragment,primers were designed so as to introduce a HindIII restriction site on the 3 end to allow ligation into thedestination vector. To ensure that these modifications were successful, the 5 and 3 fragments were ligatedinto the pGEM-T-Easy “hotel” vector and sequence-confirmed.Subsequently, the fragments were digested out. The NA5 fragment was generated by digesting with BbsI toleave the designed NcoI overhang on the 5 end and a non-specific sticky end on the 3 end. The NA3 fragmentwas generated by digesting with BamHI leaving a 5 BamHI sticky end, PCR-purifying and then digesting withHindIII to leave a 3 HindIII sticky end. The 348 bp NA5’ fragment and 306 bp NA3’ fragment were gel-isolatedand purified.Internal Insert Fragment. The internal fragment was generated by digesting parental plasmid pT7R-NoV1,which contains the NA cDNA sequence, with BbsI to leave a 5 non-specific sticky end that is complementary tothe NA5 fragments 3 sticky end, PCR-purifying and then digesting with BamHI to leave a 3 BamHI sticky end.These ends would allow the internal fragments ligation with the NA5 and NA3 fragments. The 2583 bpfragment was gel-isolated and purified.MATERIALS & METHODS
  6. 6. Vector Fragments. The expression vector, pET45b(+) was chosen as the destination plasmid. It drives transcription of agene-of-interest under the control of a T7 promoter. It contains an ampicillin resistance gene (AmpR), allowing forpropagation in selective ampicillin media. pET45b(+) was first linearized via PvuI digestion, which cuts in the middle ofAmpR. After PCR-purification, digestion with NcoI and HindIII, both of which cut in the Multiple Cloning Site (MCS),liberated two fragments of sizes 3933 bp and 1217 bp respectively. The vector fragments were gel-isolated and purified.The rationale for cutting in AmpR is that it acts to facilitate positive clone selection. Only those cells containing properlyligated plasmid with functioning AmpR will be ampicillin-resistant and grow on selective ampicillin plates.Plasmid Construction and Propagation. All five fragments were then resolved on a 1.2% TBE agarose gel and analyzed toconfirm correct size, and to ascertain purity and adequate quantity. The fragments were ligated overnight with T4 DNAligase (Invitrogen/Life Technologies, Carlsbad, CA) in the presence of T4 DNA ligase buffer (Invitrogen/Life Technologies),after which the enzyme was heat-inactivated at 60°C for 20 minutes.Transformation and Cell Culture. The final construct was propagated in NEB 10-β E. coli cells (New England Biolabs,Ipswich, MA). Cells were transformed according to manufacturers protocol. Transformed cells were amplified in 1mLSOC media (New England Biolabs) at 37°C for 1 hour, then plated on 2X Yeast Extract/Tryptone (2X YT) plates containingampicilin (10g/L Yeast Extract, 16g/L Tryptone, 5g/L NaCl [pH 7.4], 15g/L Agar, 100mg/L Ampicillin). Plates wereincubated at 37°C overnight. Ampicillin-resistant colonies were amplified in 2mL 2X YT at 37°C overnight, small-scaleplasmid preparations were performed (QIAprep Spin Miniprep Kit, Qiagen, Hilden, Germany) and positive clones werescreened by restriction profiling.DNA Gel Electrophoresis and Analysis. Running Buffer: Tris Borate EDTA (TBE) (12.1g/L Tris base, 5.1g/L Boric acid,0.37g/L Disodium EDTA [pH 8.3]), 1.2% (wt/vol) TBE agarose gel: 50mL TBE, 0.6g agarose, 25μg Ethidium Bromide(BioRad, Hercules, CA). Gels were visualized using Gel Doc XR (BioRad) automated UV transilluminator.Digests and DNA Purification. All restriction digests were set up using enzymes and appropriate buffers from NewEngland Biolabs according to manufacturer’s protocols. PCR Purification and Gel Purification Kits by Qiagen.MATERIALS & METHODS (Cont.)
  7. 7. RESULTSFig. 1: pET45b(+)-SRP-NA construction strategy. All new elements (restriction sites at 5’ and 3’ends to facilitate ligation into destination vector, start codon, and 6X His tag) were introducedinto the NA ORF at the primer design stage. Ligation into pGTE subclone vectors facilitatedsequence confirmation of the newly modified NA5’ and NA3’ fragments.Purpose of each element: The SRP (Signal Recognition Particle) element will target proteinsynthesis at the E. coli cell membrane allowing us to obtain soluble protein using membrane-solubilization methods. The crucial introduction of the 6X His tag on the 5’ end of the SRP-NAORF will allow for Ni2+-NTA affinity purification of the recombinant protein. Cleavage with FactorX at the advantageously positioned Factor X Cleavage Site (Fac X) will remove NA from thebound His-tag yielding pure wild-type NA.Note on the generation of vector fragments: The destination vector was first cut in the middleof the ampicillin resistance gene (AmpR) with PvuI and then with NcoI and HindIII to yield thetwo vector fragments. This cut in the AmpR gene acts as a measure to further facilitate theselection of positive clones; only those cells that are transformed with properly ligated plasmid,containing a functioning AmpR gene, will grow on selective ampicillin plates.
  8. 8. Fig. 2: TBE agarose gel electrophoresis ofDNA fragments. Cloned DNA fragmentsfor pET45b(+)-SRP-NA construction wereanalyzed for size, purity and quantity on a1.2% TBE agarose gel. Lanes:1. 1 kbp ladder2. pET45b(+) [PvuI-NcoI] vector fragment(3933 bp)3. pET45b(+) [PvuI-HindIII] vectorfragment (1217 bp)4. pT7R-Nov1 [BbsI-BamHI] NA internalfragment (2583 bp)5. 100 bp ladder6. pGTE-NA5’ [BbsI(NcoI)-BbsI] NA5’fragment (348 bp)7. pGTE-NA3’ [BamHI-HindIII] NA3’fragment (306 bp).Gel was visualized on a Gel Doc XR(BioRad). All fragments are of desiredsize, purity and quantity.1 2 3 4 5 6 7
  9. 9. Fig. 3: Final constructpET45b(+)-SRP-NA.Restriction sites usedto ligate insertfragments into vectorare shown. Ampicillinresistance gene(AmpR) enablespropagation ofpositive clones onselective media.Transcription ofrecombinant SRP-NAis under control of T7promoter. Also, originof replication (Ori) isshown.pET-45b(+)-SRP-NA(8380 bp)BbsIBamHIHindIIINcoIAmpROriT7 Promoter6xHis-SRP-FacX-Nov RdRp
  10. 10. Endogenous InduciblePlasmidpET45b(+)-SRP-NAT7 RNAPolIPTGRNASRP X NA6XHisFactor XCleavageSitepET45b(+)-SRP-NATransformCell MembraneT7 EXPRESS E. coli(NEB)FUTURE WORK
  11. 11. Fig. 4: Protein Induction. T7 EXPRESS E. coli will be transformed with thenewly constructed plasmid, pET45b(+)-SRP-NA and grown in culture to anoptimal optical density. The cells, which carry endogenous plasmidencoding T7 polymerase under regulation of the lac operon, will beinduced with isopropyl-β-D-thio-galactoside (IPTG) to synthesize highlevels of the T7 polymerase. This will drive transcription of therecombinant SRP-NA, which is under control of a T7 promoter.Synthesized protein will be targeted to the cell membrane via the SRP-SecY pathway due to the presence of the N-terminal SRP signal. Proteinwill then be harvrested.FUTURE WORK
  12. 12. We will transform competent T7 EXPRESS E. coli (New England BioLabs) cellswith the newly cloned pET45b(+)-SRP-NA. We will use individual colonies toinoculate 2X YT/Ampicillin cultures and allow growth until the optimal opticaldensity for protein induction. We will induce expression of the recombinantprotein with IPTG (3). The SRP tag will target protein expression to the cellmembrane (6). Subsequently we will follow a lysis procedure from which we willharvest the membrane fraction via ultracentrifugation. The membranes will besolubilized using detergents to release the recombinant protein in soluble form.The solubilized membrane proteins will be loaded onto a Ni2+-NTA column andpurified via affinity chromatography. The 6X His tag will enable binding onto thecolumn. After several washes, on-column cleavage of the NA protein will beperformed with FacX to obtain purified protein (3).The purified NA will then be used to conduct in vitro studies on the replicationinitiation mechanism of the NoV RNA-dependent RNA-polymerase (3,4,5). It willalso be used to obtain a crystal structure.
  13. 13. REFERENCES1. Ball, L.A., Amann, J.M., and Garrett, B.K. 1992. Replication of Nodamura virusafter transfection of viral RNA into mammalian cells in culture. Journal ofViroogy. 66:2326-2334.2. Johnson, K.N., Johnson, K.L., Dasgupta, R., Gratsch, T., and Ball, L.A. 2001.Comparisons among the larger genome segments of six nodaviruses and theirencoded RNA replicases. Journal of General Virology. 82:1855-1866.3. Lai V.C.H., Kao C.C., Ferrari E., Park J., Uss A.S., Wright-Minogue J., Hong Z., LauJ.Y.N. (1999). Mutational Analysis of Bovine Viral Diarrhea Virus RNA-Dependent RNA Polymerase. Journal of Virology. 73: 10129-101364. Ranjith-Kumar, C.T., Kim Y., Gutshall L., Silverman C., Khandekar S., Sarisky R.T.,Kao C.C. (2002). Mechanism of De Novo Initiation by the Hepatitis C virus RNA-Dependent RNA Polymerase: role of Divalent Metals. Journal ofVirology.76:12513-125255. Sun J.H., Adkins S., Faurote G., Kao C.C. (1996). Initiation of (–)-Strand RNASynthesis Catalyzed by the BMV RNA-Dependent RNA Polymerase: Synthesis ofOligonucleotides. Virology. 226:1-126. Valent Q.A. (2001). Signal recognition particle mediated protein targetting inEscherichia coli. Antoine van Leeuwenhoek. 79:17-31
  14. 14. We thank the UTEP BBRC DNA Analysis Core Facility. Thisproject was supported by grant number 5G12RR008124 fromthe National Center for Research Resources (NCRR), acomponent of the National Institutes of Health (NIH). Wewould especially like to thank Ana Betancourt for DNAsequencing.ACKNOWLEDGEMENTSCloning and expression of the Nodamura virus RNA-dependent RNA polymerasePoster presentation at Society for the Advancement ofChicanos and Native Americans in Science (SACNAS)National Conference, October 2012, Seatltle, WA