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Newcastle disease is caused by specified viruses of the avian paramyxovirus type I (APMV-I) serotype of the genus Avulavirus belonging to the subfamily Paramyxovirinae, family Paramyxoviridae. Since its recognition in 1926, ND is regarded as being endemic in many countries (Alexander, 1997). NDV has been shown to be able to infect over 200 species of birds, but the severity of diseased varies with both host and strain of virus. Strains of NDV have been grouped into five pathotypes on the basis of the clinical signs seen in infected chicken, which are; asymptomatic enteric, lentogenic, mesogenic, neurotrophic velogenic and viscerotropic velogenic strain. Since this disease has become an ongoing threat to the poultry industry, the development of a better diagnostic kit is required and can be achieved through selecting an immunogenic part of virus that could produce a suitable antigenic response.
Several commercials kit for NDV specific antibodies detections are already available, which used inactivated virions as coating antigen. However, the propagation and purification of NDV is tedious, unsafe and expensive. Compared to full virions, recombinant protein produce through gene engineering detect only specific antibodies to one certain protein and are safer, easier to produce and less expensive. Nucleocapsid protein of NDV has been recognized an immunogenic and produced an immune response in chicken.
Currently, the difficulty in obtaining large quantity of viral antigen is the main barrier in the production of diagnostic kits. NP protein of NDV has been expressed in Baculovirus (Errington and Emmerson, 1995) and E.coli expression system (Kho et al., 2001). However, expression of proteins in such system would not produced desirable amount to use in downstream application. Yeast expression system become an alternative host for researcher even manufacturer to produce heterologous protein. This system combined the advantages of prokaryotic and higher eukaryotic expression system. Nowadays, Pichia expression system was fully employed for production of desired protein, either in laboratory or industries.
Pichia pastoris has many advantages as an eukaryotic expression system for recombinant proteins, including multiple copy selection, strong promoter activity, high yield production of desire protein, and facilitation of secretion. This system is particularly suitable for the production of proteins that form inclusion bodies in E.coli, and whose expression levels are very low in mammalian cell lines. Nowadays, this system was widely employed by researcher even a biotechnology companies for the production of vaccines, antigens, antibodies, hormones, protease inhibitor and ligands. A commercial kit based on P.pastoris also available and subject to further improvement. At the same time, fermentation process optimization is likely to make this system more competitive, and reproducible in producing relevant compounds on libratory or industrial scales.
Material and method
Strains and media
Escherichia coli TOP10 [F- mcrA Î”(mrr-hsdRMS-mcrBC) Ï†80lacZÎ”M15 Î”lacX74 nupG recA1 araD139 Î”(ara-leu)7697 galE15 galK16 rpsL(StrR) endA1 Î»-] (Invitrogen, USA) was used as the host strain for amplification and propagation of recombinant DNA. E.coli was cultured at 37 oC in low salt LB medium (0.5% yeast extract, 1% NaCl, 1% tryptone) and supplemented with 25 ug/ml Zeocin (Invitrogen, USA). P.pastoris GS115 (his4) strain was purchased from Invitrogen (USA). Yeast was grown at 30 oC on rich YPD medium (1% yeast extract, 2% peptone, 2% glucose) and MMH medium (1.34% YNB, 0.00004% biotin, 0.5% methanol).
Construction of expression plasmid
A NP gene was amplified by polymerase chain reaction (PCR) from the plasmid vector pTrcHis2-NP (Kho et al., 2001). PCR was carried out using the following oligonucleotides primer: FP (5'- ACA CGA ATT CAT GTC TTC CGT ATT CGA TGA-3') and RP (5'-GTC TCT CGA GTC AAT ACC CCC AGT CGG TGT -3') containing EcoR I and Xho I site (indicated in bold). The condition of PCR was followed: template was initially denature at 94 oC/5 min followed by 33 cycles (denaturation at 94 oC/ 1 min, annealing at 68 oC/ 1 min, elongation at 72 oC/1 min) and final extension was performed at 72 oC for 10 min. The PCR products were cut off by EcoR I and Xho I and the fragment were ligated into the multiple cloning site of digested yeast expression vector, pPICZ A. The resulting expression vector was named pPICZA/NP. The insertion of NP gene was confirmed by PCR and restriction enzyme digestion.
Transformation of P.pastoris, selection of Mut+ and multi-copy transformant
Transformed DNA was linearized using Pme I leading to targeting of recombinant plasmid to AOX I locus. Ten micro litters of pPICZA/NP was added to 80 ul of P.pastoris competent cells and transferred into a 2 mm gap electroporation cuvette () that was pre-cooled on ice for 5 min. P.pastoris strain was transformed by electroporation at 1.5 kV, 200 Î© and 25 uF for 5 ms using a Gene Pulser II system (Bio-Rad,USA). Immediately after the pulse, 1 ml of cold 1M Sorbitol was added and the suspension was grown for 2 h at 30 oC without shaking. Aliquots of 150 ul were spreaded onto YPD plates containing 100 ug/ml of Zeocin and incubated for 3 day or until colonies appeared at 30 oC.
Using a sterile cotton-swab, the colonies which appeared on 100 ug/ml Zeocin plates were picked and streaked in a regular pattern on MMH and MDH media plates and incubated at 30 oC for 2-3 days. The His+Mut+ transformants were differentiated from His+Muts through comparison of patch growth rate on MMH and MDH plates. It was followed by selecting all the Mut+ phenotype and streaked on the YPDS media containing different concentration of Zeocin (500 ug/ml, 1000 ug/ml and 2000 ug/ml) in order to determine multi-copy insertion. The plates were incubated at 30 oC for 2 days. The colonies which have the fastest growth were chosen for protein expression.
PCR analysis of P.pastoris recombinant transformant
The genomic DNA of P.pastoris recombinant transformant was isolated as described by Ayra Pardo et al., (1998). PCR amplification was performed as followed: initial denaturation at 94 oC for 4 min, pursued by 30 cycles (each at 94 oC for 1 min, 68 oC for 1 min and 72 oC for 75 s) and a final extension at 72 oC for 10 min. The genomic DNA s isolated from recombinant P.pastoris transformed with parent plasmid was used as control in PCR.
SDS-PAGE and Western blotting
Samples were boiled in reducing sample buffer was run in SDS-Tris-glycine buffer (pH 8.8). Proteins were stained with Coomassie brilliant blue. After SDS-PAGE electrophoresis proteins were transferred to PVDF membrane. The blots were blocked with 5% milk in PBS for 30-60 min. The blocking solution was removed and the blots were incubated with polyclonal antibody, anti-NP (1: 5000). The membrane was then thoroughly washed with TTBS washing solution and exposed to alkaline phosphatase antibody for 1 h. The membrane was then washed again thoroughly before BCIP/NBT (5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium) solution was added as substrate for the phosphatase.
Optimization of P.pastoris protein production
The induction conditions for the best NP expression levels and quality were chosen by optimizing methanol concentration and dissolved oxygen in shake flask. Before optimization procedure, time-course study was performed in order to determine the time point that yielded the highest concentration of NP protein. Protein amounts were determined using the Bradford method. To investigate the effect of methanol concentration on NP expression, induction was performed in MMH medium containing 0.5, 0.75, 1.0 and 1.5% methanol using 50 ml culture in 250 ml shake flask with an inoculums density of OD600 = 1.0. The effect of dissolved oxygen in media on NP protein expression was also investigated. Different volume media (10, 20, 30, 40 and 50 ml) were loaded in 100 ml identical shake flask. The amount of methanol was added after the best yielded time point and the higher result on final concentration of methanol was determined. The NP expressions using the two factors at the optimal time were analyzed by SDS-PAGE, Western blot and Bradford assay to determine the optimal induction conditions.
Scale up of expression
After optimization, MGYH medium (50 ml) in 250 ml shake flask was inoculated with well isolated colony. The cell were grown at 30 oC in a shaker (300 rpm) until the culture reached an OD600= 2-6. This 25 ml culture was centrifuged and used to inoculate 100 ml of MGYH in 1-l shake flask, and the culture was grown at 30 oC with vigorous shaking (300 rpm) until reaching OD600= 2-6. The cells were harvested in sterile falcon tube by centrifuging at 4000 rpm for 5 min at room temperature. To induce the expression, the supernatant was decanted and the cell pellet was resuspended to the 1.5 inoculum density in MMH at the optimal methanol concentration and time point of induction. Five hundred milliliters of the culture were divided between several 1-l baffle flasks. The flasks were covered with two layers of sterile gauze, returned to incubator, and the cultures allow growing at 30 oC with shaking. Methanol was added to the optimal concentration every 24 h until the optimal time of induction, as determined from time course study, was reached. The yeast cells were removed by centrifugation at 4000 rpm for 15 min and store at -80 oC until its ready for further analysis.
Purification of NP protein
For the purification, the yeast culture was resuspended in breaking buffer [50 mM Sodium phosphate (pH 7.4), 1 mM PMSF, 1 mM EDTA and 5% glycerol] containing 1 mM PMSF and disrupted mechanically in the present of glass beads. The yeast expressed NP protein was purified by successive ultracentrifugation through 10-50% sucrose cushion (38 500 rpm, 3 h). Fraction containing proteins were identified by SDS-PAGE. After first centrifugation in sucrose gradient fractions containing protein with the molecular weight corresponding to NDV nucleocapsid protein (53 kDa) were pooled, dialyzed against dialysis buffer (50 mM Tris, 100 mM NaCl; pH 7.8). The dialyzed solution was applied to another 10-50% sucrose gradient and centrifuged at 38 500 rpm for 3 h at 4 oC. Fraction containing NP protein were pooled together and concentrated with a 10 kDa cut-off Centricon centrifugal filter (Milipore, USA). The concentration of purified NP protein was determined using Bradford assay.
Results and discussion
Amplification of NP gene and construction of pPICZA/NP recombinant vector
An approximately 1500 bp fragment of NP gene of NDV strain AF2240 was successfully amplified through PCR (Fig.1) using FP and RP primers. After EcoRI and XhoI digestion, purification and ligation to pPICZA, the construct was used to transform the E. coli Top 10. The plasmid extracted from Zeocinâ„¢-resistant transformants was digested with EcoRI and XhoI restriction enzyme was resulted 3300 bp and 1500 bp fragment were obtained as expected (Fig. 2, lane 2). This expression plasmid was designated pPICZA/NP. Sequencing showed that the open reading frame of NP gene was 96% homology with nucleotide sequence of NP gene (NDV strain AF2240) obtained from NCBI (accession no. AF284646).
Transformation of P.pastoris
The constructed pPICZA/NP plasmids were linearized with the restriction enzyme PmeI to allow integration of the vector into the chromosomal DNA of P.pastoris strain GS115. The linearized plasmids were introduced into the competent yeast cell by electroporation. The transformants were selected on YPDS plates containing 100 ug/ml Zeocin. Fifteen clones were number and streaked on MM plates, MD plate and on set of Zeocin YPDS plates (Zeocin concentration ranging from 0.5-2.0 mg/ml). One of the clones, which were resistant to the higher concentration of 2.0 mg/ml Zeocin and grew on MM and MD plates, was subjected to further selection for a small scale expression. In order to determine expression kinetics, the clone was subjected to small scale expression and culture was collected at 0, 12, 24, 36, 48, 60 and 72 h after induction using SDS-PAGE analysis. From the result (Fig), we noted that the NP protein was successfully expressed in this methylotrophic yeast system. In order to confirm the expression, Western blotting procedure was performed using anti-NP primary antibody. The result revealed that the NP protein was expressed after 12 h of induction with the correct molecular weight of 53 kDa. The concentration of the protein expression was achieved using Bradford assay. It is important to know the highest level of protein expressed before methanol induction phase is optimize. Result in the table demonstrated that induction of methanol after 24 h gave higher concentration which is 0.29 mg/ml. The expression level of NP protein decreased gradually after 36 h and the lowest concentration was detected at 0 h which is 0.01 mg/ml. This result showed that there is no expression of protein during repression stage (glycerol culture) and the protein can be expressed just only if the methanol was fed to the Pichia culture. Pichia is methanol fed yeast system and need a methanol as a carbon source.
Optimization of Pichia protein expression
Expression levels of foreign protein depend not only on the native sequence of cDNA, such as codon bias, but also on other factors, such as copy number, temperature, pH, induction duration, and concentration of inducer (D'Anjou and Daugulis, 2000. M.C. D'Anjou and A.J. Daugulis , A rational approach to improving productivity in recombinant Pichia pastoris fermentation. Biotechnol. Bioeng. 72 (2000), pp. 1-11.D'Anjou and Daugulis, 2000). In the present study, the final concentration of methanol and dissolved oxygen had already been optimized. The optimum process parameters for the expression of NP protein are (a) an induction time of 24 h, (b) a methanol concentration of 1.0% and (c) dissolved oxygen ranging to 10-20% of shake flask volume.
The above study was limited to shaking-flask culture, and production could be expected to be about 10 fold higher by high-density fermentation, because P. pastoris is a yeast well suited for fermentation (Cregg et al., 1993). It is evident that future work is necessary to investigate the optimal conditions for recombinant NP protein production in large-scale fermentation.
Under optimal condition, the yield of NP from the scale-up of expression with GS115/NP was 571 mg/l, which is at least 49.21% higher than that from un-optimized condition in P.pastoris (290 mg/l). The result demonstrate that optimization of P.pastoris protein production can increase yield of protein production. Moreover, because purification P.pastoris simply requires an initial concentration of the medium in conjunction with high expression level, this organism is an attractive expression system. The ease of the expression in its cells and purification of NP protein from P.pastoris confirms the potential of this system for producing large quantities of protein needed for downstream application.
As regards the expression in P.pastoris, it is well known that the yeast cell wall is thick and very resistant. The recommended lysis technique is using a glass bead beater (D.Rolland et al., 2001).
We can suggest that the density gradient centrifugation could induce deformation and aggregation of virus-like particles, due to over concentration or dehydration of particles in high sucrose density resulting protein degradation.
The lower methanol concentration limits cell growth due to carbon limitations and the higher methanol concentration is toxic because accumulation of its metabolic products (Cregg et al., 1993). Figure X showed that, after incubation for 24 h, a methanol feed of 1% (v/v) led to the highest volume of NP protein expression and a methanol feed of 1.5% (v/v) led to the lowest expression.
For expression of proteins using the AOXI promoter, it is important to always keep the methanol level within a relatively narrow range (cereghino and Cregg, 2000).