CLONING AND EXPRESSION OF Lb-PROTEASE FROM cDNA CLONE OF FOOT-AND-MOUTH DISEASE VIRUS

Foot-and –Mouth disease virus (FMDV) is a positive sense RNA virus and the genome codes for single polyprotein. The FMDV L protein is located at the N terminus of the polyprotein and is the first gene product released from the nascent polyprotein. The leader L protease which is involved in pathogenesis has two known functions: (i) auto-catalytic removal from the N terminus of the viral polyprotein and (ii) cleavage of the p220 subunit of the eukaryotic initiation factor 4F complex, which helps to shut off host protein synthesis. To explore the role of L protease in FMDV pathogenesis we generated synthetic FMDV genome lacking the L gene. The gene was amplified from an infectious cDNA clone of serotype Asia1. Primers corresponding to L protease were designed based on the sequence available in the data base. An amplified DNA of 546bp was purified and cloned into pET28 cloning vector. The sequence analysis revealed the presence of single Open Reading Frame (ORF) encoding a protein of 173 amino acid residues. The sequence alignment using BLAST search in NCBI gene Bank showed 91% homology with FMDV strain A isolate IND17/77 L protease gene. The recombinant plasmids pETLb was transferred into BL21 (DE3) pLysS cells and the IPTG induced expressed protein of 25 KDa was purified by nickel affinity column as per the manufacturer’s protocol (Sigma, USA). The specificity of the expressed protein in was confirmed by western blotting using convalescent cattle serum/ rabbit anti-bovine horse radish peroxidase conjugate and O-Dianisidine Dihydrochloride substrate.


Introduction
Foot-and-mouth disease (FMD) is one of the most contagious diseases of cloven hoofed animals having high economic impact.The causative agent, a virus belongs to the genus Apthovirus, family picornaviridae [1].FMD is an economically devastating disease of livestock and the presence of the virus in part of the globe is a threat to rest of the world.Although mortality is usually low, morbidity can reach 100% causing severe losses in production.Therefore, the disease remains a major concern to many developing countries and a continued threat to disease free countries [2,3].Millions of animals are sacrificed every year worldwide under FMDV eradication programmes [4].FMDV has continuously circulated ever since after the first outbreak in America in 1870 [5,6].Further, new serotypes are continuously evolving due to an infinite mutation rate in the RNA genome of the virus [7].Currently vaccination in India is carried out using trivalent inactivated viral vaccines but there are several limitations associated with inactivated vaccines, major of which is the release of live virus either while handling or through the vaccine, propagation of virulent virus, limited shelf-life and booster injection requirement after 4-12 months [8].Under this situation production of attenuated virus based vaccine may be safe and ideal.Attenuated virus vaccines are time tested for having proven their worth in eradicating many diseases.However, being highly mutable, naturally selected FMDVs are not considered as reliable vaccine candidates.With the availability of advanced genetic engineering techniques it is now possible to delete any gene segment and make the virus replication defective.Such viruses are ideal candidates for development of attenuated virus vaccines.The FMDV genome contains a single open reading frame of about 7000 bases in length that is initiated from an internal ribosome entry site (IRES) about 1000 bases from the 5' end of the RNA.The Leader (L) appears to cleave at only a single site in the poly protein.The L gene is positioned at the 5' end of the open reading frame and contains two potential in-frame initiation codon(84 nucleotides apart).The L-Protease is responsible for pathogenicity by inhibiting the host protein synthesis.L-Protease mutants have been found to reduce pathogenicity and replication efficiency of the virus.Moreover the deletion helps in developing marker vaccine.
Here we report the cloning, expression and characterization of L-Protease.The gene is expressed in E.coli and the protein expressed is used to raise antiserum in rabbit which could be further used to develop companion tests for evaluating marker facility of the gene deleted attenuated virus.

Report
Gene, Host strain and Plasmid Vectors: Foot and mouth disease virus serotype Asia 1 63/72 infective cDNA clone available in the laboratory (Suryanarayana et al 2003) was used for amplifying Lb-Protease gene.Commercially available E.coli host strains DH5α and BL21 DE3 p(Lys)S and expression vector pET 28 A were used for L-protease expression.

Lb protease amplification Cloning:
The Lb-Protease sequence was amplified from the full length infective cDNA using the primers LPro (L) 5' GCG CCA TAT GGA ATT CAC ACT CTG CAA CGG T as forward and L-Pro (R ) GCG CTC GAG TGC GGC CGC CTT GAG TCG TTT CTG AAC CTT as reverse primers.These primers were designed based on the published sequence (Saravanan et al 2003).A 50µl reaction mix containing 1.5mM Mgcl 2 , 100µM each of dNTPS, 25mM Tris-HcL, 20 pmol of each primer and one unit of Taq DNA polymerase was used for amplification in the Thermal Cycler (perkin-Elmer Cetus, USA) with initial denaturation at 95°C for 3min, followed by 35 cycles of 95°C for 1min, 60°C for 1min and 72°C for 1min with a final extension at 72°C for 10min.the amplified PCR product corresponding to Lb-Protease (500bp) gene was purified using wizard PCR prep DNA purification system (Promega, USA), digested with Nde1 and Xho1 and the digested product was ligated into pET28 at Nde1 and Xho1 sites.The transformants were selected on kanamycin plates.The transformants were initially screened by colony PCR, using vector specific T7 promoter sequence primer and insert specific reverse primer to select the recombinants, followed by insert release from the recombinant plasmid DNA.

Sequencing of Lb-Pro Gene:
The L-Pro gene in the plasmid pETLb was sequenced using T7 promoter and Lpro(R) primers in ABI 377 automated DNA sequencer (ABI inc., USA).Sequence data obtained was analyzed by aligning with published FMDV L-protease gene sequences available in NCBI data base.Both nucleotide and derived amino acid sequence were compared with the published Asia 1 sequence.

Expression and purification of cloned Lb-Pro:
The recombinant plasmids pETLb was transferred into BL21 (DE3) pLysS cells.Three individual clones were selected and grown seperately in 10ml LB broth with Kanamycin(50µg/ml) for overnight at 37°C with shaking.One hundred microliter of the overnight culture was inoculated into fresh 10mL LB broth without antibiotic for 3 h at 37⁰C.The protein expression was induced by adding IPTG (isopropyl-beta-D-thiogalactopyranoside) to a final concentration of 1mM at 30⁰C for 6 h and the cells were pelleted and resuspended in 1mL of TE containing 10mMphenyl methyl sulfonyl fluoride ( PMSF) (pH 8.0) and stored at -80⁰C for further work.The expressed protein in the cell lysate was purified by nickel affinity column as per the manufacturer's protocol (Sigma, USA).The proteins in the cell lysate were diluted 4 folds with column loading buffer (8M urea in 10 mM Tris HCl, 1mM EDTA, pH.8.0) and centrifuged at 15000xg at 4 0 C to remove any debris.The solution was concentrated by polyethylene glycol, dialysed against phosphate buffered saline (PBS) and stored at -70⁰C.The purified protein was separated on 12% polyacrylamide gel under denaturing conditions as per the mothod of Laemmli.
An aliqot (20 ul) of the cell lysate was analysed by 10% Sodium Dodecyl Sulpfate-Polyacrylaminde Gel Electrophoresis (SDS-PAGE) as per standard protocol in comparison with the same quantity of lysates from vector transformed cells and uninduced cloned cells.The specificity of the expressed protein in was confirmed by western blotting using , the convalescent bovine serum as primary antibody, and antibovine antibody horse radish peroxidase conjugate as secondary antibody and O-Dianisidine Dihydrochloride as substrate as per standard protocol (Towban) A similar duplicate gel was blotted onto a PVDF membrane for immune detection.The recombinant protein was detected by treating with anti-convalescent serum (at 1:1000 dilution), followed by an anti-rabbit antibody HRPO conjugate (1:1000) and orthodianisidine dihydrochloride (ODD) as substrate.

Results and Discussion
The amplified PCR product corresponding to L-Protease b (Lb) of size 546 nts.(519 gene size + 27nts (Fig. 1) was purified using wizard PCR prep DNA purification system (Fementas, USA) and cloned into Nde1 and Xho1 sites in frame in pET28a vector.The cloned insert was subjected to nucleotide sequence using T7 promoter primer as described under Methods.A sequence of 743 nts was read from the ladder sequence from 5' end which includes vector sequences (Fig. 3).When the nucleotide sequence was translated using on-line programme an aa sequence of 173 aa (Fig 4) was obtained with no stop codon in between.Identity of the cloned sequence was confirmed by Nucleotide Blast search using NCBI online programme of sequence analysis (Fig. 5)  The BLAST search (Fig 5 ) showed the presence of FMDV L protease gene sequence devoid of 87 nucleotides corresponding to 29 aa at N-terminal end, termed as Lb protease.As shown in Fig. 5, when the ladder sequence was aligned with the L-protease gene of FMDV 'A' the alignment shows that the protein has a homology of 91% with the published L-protease gene sequence.The second ATG codon is in frame with the rest of the sequence indicating the intactness of the second coding region which is reported to be in-frame with the fist ATG codon.The protein derived from second ATG codon is designated as Lb while entire protein is designated as Lab.As per the literature both are active proteins and can function Cis-active proteases, which are released post translational from poly protein at N-terminal end of the FMDV poly-protein of 250 kDa.Preference for initiation codon depends on Mn 2+ concentration.The recombinant pET Lb was transferred into BL-21 DE3 pLys cells.Three of the several transformants were selected, grown in LB broth in the presence of Kanamycin and induced with IPTG for expression of the cloned gene and the lysate proteins were analyzed by 10% SDS SAGE.The gel was stained with Comassie Brilliant Blue (CBB) and de-stained as described under materials and methods (Fig 6).As seen in the Fig 6 Several protein bands were seen in the CBB stained gel in the case of both induced and un-induced cells (lane 1-6) which may be host or vector specific.However, a single intense protein band of size around 25 kDa is visible only in the case of induced cultures carrying vector with Lb gene as insert (lane 1-3) but not in the case of un-induced (lane 4,5) and vector transformed and induced culture (lane 6) indicating that 25 kDa protein corresponds to the cloned Lb protein gene.The other proteins may be of host or vector specific.carring N-Terminal (20aa) and c-terminal (13aa) fusion that has all the Tags including His Tag.By inserting the gene lacking termination codon at 3' end, the His Tag at the N-terminus of the vector is also in frame.Therefore the total coding region excluding Tag protein comprises 645 nucleotides i.e 215 amino acids corresponding to ~24KDa.High intensity protein bands revealed that the protein was over expressed and present in soluble form.
The protein corresponding to Lb-Protease as per the sequence was expected to have molecular weight of 19 KDa.However the observed molecular weight of 24KDa is due to the presence of tags at Nand Ctreminal regions.Presence of His-tag at amino terminus of expressed proteins from pET28a vector had enabled us to purify the recombinant protein from E.coli using HISselect nickel affinity gel column (Fig. 7).The specificity of the expressed protein was confirmed by Western Blot assay where antiserum from convalescent bovine was used.The intense positive colour reaction shows the specificity (Fig. 8).The background colour reactions seen above and below the specific protein may be due to the presence of other E.coli proteins which have co purified as contaminants that were not visible in the CBB stained gel (Fig 8 , lane 1).  the N terminus of the viral polyprotein and cleavage of the p220 subunit of eIF-4F.Cleavage of p220 is thought to be responsible for inhibiting the translation of capped host cell mRNAs in the presence of viral translation which occurs by a cap-independent mechanism.Cleavage of p220 appears to be an important for picornavirus replication, since the rhinoviruses and enteroviruses utilize a different proteinase (2A) to cleave p220.
The major objective of producing L protease is to raise antiserum against L-Pro and utilize the same for the detection of attenuated leaderless virus developed by us.The L-proteinase gene of FMDV was amplified and sequenced.The complete L proteinase gene could be expressed in soluble form with six histidines which was used as a tag to purify the soluble protein expressed in E.coli.The specificity of the expressed protein was confirmed by Western Blot assay blot assay where antiserum against Lb-Protease (purified E.coli expressed protein) raised in rabbits was used.The calculated size of the L-Protease comes to 24KDa which is in agreement with the protein size observed in SDS-PAGE.The protein could be used as diagnostic antigen for the development of companion test to be used in L-deleted attenuated FMDV vaccine.

Figure 5 :
Figure 5: Sequence alignment of the ladder sequence using BLAST search programme

Figure 6 :Lane 1 , 2 , 3 .
Figure 6: SDS PAGE anlysis of E.coli expressed proteins from pET 28 vector with or without interest

Figure 7 :
Figure 7: SDS PAGE of the affinity purified Lb protein expressed in E.coli

Vector sequences are underlined. His tag sequences at 5' and 3' ends from the vector are shown with italics. ATG codons are shown with bold letters.
[Sarkar