Heterologous expression of βC1 of chili leaf curl virus in pichia pastoris

Chili pepper is naturally susceptible to a wide range of viruses in all south Asian countries including Pakistan. Chili leaf curl virus is a monopartite begomovirus having single stranded circular betasatellite. It has one open reading frame βC1, required for pathogenicity determined, symptom induction and viral accumulation. It produces viral symptoms like mosaic, mottling, leaf distortion, vein etching, yellowing, stunting and narrowing of leaves. This study was conducted on the basis of βC1 protein, whether it was expressed in prokaryotic and yeast expression system or not because many viral proteins are lethal for the host organism. For this study, specific set of primers for βC1 were designed and amplified product was inserted into pET32a(+) bacterial and pPIC3.5K Pichia vectors for its expression. βC1 was not expressed in BL21 Escherichia coli expression system, while it was expressed in Pichia pastoris , when it was integrated into the genome through electroporation, and expressed protein was identified by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDSPAGE). This is the first study demonstrating the possibility of expression of βC1protein using P. pastoris . Key words : Monopartite begomoviruses, chili leaf curl betasatellite , heterologous expression, Pichia pastoris , betasatellite.


INTRODUCTION
Chili pepper is naturally susceptible to a wide range of viruses in all south Asian countries including Pakistan, producing various symptoms like mosaic, mottling, leaf distortion, vein etching, yellowing, stunting and narrowing of leaves (Figure I).So far, 66 viruses have been reported infecting pepper worldwide (Green and Kim, 1991).In Pakistan and some other parts of the world, ChLCB and CMV are the major viruses prevalent in chili growing areas and reducing yield up to 60, 50 and 40%, respectively (Shah and Khalid, 1999).*Corresponding author.E-mail: drsaiqa@gmail.com.
Among these, ChLCB is the most important pathogen associated with chili crop (Shah et al., 2001).
Chili leaf curl virus belongs to the begomoviruses family, Geminivirdae.ChLCB is a monopatite begomoviruses having a novel type of circular single stranded satellite DNA, referred to as betasatellite.It has recently been found to be associated with some of the viruses and is required for symptom induction in plants.Betasatellite molecules are approximately 1350 nt in length (about half that of the genomes of their helper viruses) on which it depends for its replication, encapsidation, insect transmission and movement in the host plants.Numerous economically important diseases and even the earliest recorded plant viral diseases are now known to be caused by, or associated with the begomovirus/ betasatellite complex (Mansoor et al., 2003).
Pichia pastoris is a methylotrophic yeast, capable of metabolizing methanol as its sole carbon source.Methanol is converted into formaldehyde, this reaction generates hydrogen peroxide.To avoid hydrogen peroxide toxicity, methanol metabolism takes place in the specialized cell organelle, called peroxisome.Alcohol oxidase has poor affinity for oxygen and P. pastoris compensates by generating large amount of the enzyme.The promoter regulating the production of alcohol oxidase is the one used to derive heterologous protein expression in Pichia.
The P. pastoris heterologous gene expression system has been utilized to produce attractive levels of a variety of intracellular and extracellular proteins of interest.Recently, advances had improved its utility and these include: (1) methods for the construction of P. pastoris strains with multiple copies of AOX1-promoter-driven expression cassettes; (2) mixed-feed culture strategies for high foreign protein volumetric productivity rates; (3) methods to reduce proteolysis of some products in high cell-density culture media; (4) tested procedures for purification of secreted products; and (5) detailed information on the structures of N-linked oligosaccharides on P. pastoris secreted proteins (Cregg et al., 1993).
The P. pastoris expression system is being used successfully for the production of various recombinant heterologous proteins.Recently, developments with respect to the Pichia expression system have had an impact on not only the expression levels that can be achieved, but also the bioactivity of various heterologous proteins.Macauley et al. (2005) reviewed some of these recent developments, as well as strategies for reducing proteolytic degradation of the expressed recombinant protein at cultivation, cellular and protein levels.The problems associated with post-translational modifications performed on recombinant proteins by P. pastoris were discussed, including the effects on bioactivity and function of these proteins, and some engineering strategies for minimizing unwanted glycosylations.Cregg (2007) illustrated the construction of P. pastoris expression strains, the general growth and manipulation of this yeast expression system in many ways similar to those of bacterial expression systems, particularly Escherichia coli.To aid in preventing P. pastoris users from falling into one or more or these traps, this introduction focuses directly on key ways on which the P. pastoris expression system is different.Daly and Hearn (2005) studied the different features and developments under the influences of P. pastoris strain selection; the choice of expression vectors and promoters; procedures for the transformation and integration of the vectors into the P. pastoris genome; the consequences of rare codon usage and truncated transcripts and techniques employed to achieve multi-copy integration numbers.
This research work was conducted in National Institute for Biotechnology and Genetic Engineering (NIBGE), Pakistan in molecular virology and gene silencing laboratory.In this research paper, the expression of βC1 (ChiLCV) in P. pastoris (yeast) expression system is described.

Cloning of βC1 of Chili leaf curl betasatellite in Bacterial expression vector
A specific set of primers ChβC1pETF: GCGAATTCATGCACCA CGTATATGAATTATGTCC having EcoRI and ChβC1pETR: GCAA GCTTTCACACACACACATTCGTACATAC having HindIII was

Transformation of βC1 of ChLCB in bacterial BL21 strain
The recombinant clone pSAβC1pET32a (+) was transformed into E. coli BL21 competent cells through heat shock method.Transforments were selected on LB agar medium supplemented with specific antibiotics.Colonies were picked from the plate and used for polymerase chain reaction (PCR) directly by using specific set of primers.Composition of the reaction mixture and PCR profile indicated in PCR was confirmed by running on a 1% agarose gel.

Expression of βC1 of ChLCB in bacterial BL21 strain
Expression of pSAβC1pET32a (+) was confirmed through sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and western blotting techniques.The colonies were cultured in LB liquid medium supplemented with ampicillin (100 mg/ml) at 37°C overnight.Next day, 1 ml of overnight culture was transferred to 2 ml LB broth medium with ampicillin and grown for further 3 h at 37°C.After three hours of growth, 1 mM isopropyl β-D-1thiogalactopyranoside (IPTG) was added which acted as an inducer.The culture was incubated at 37°C in a shaker and allowed to grow for 8 h.Samples were taken out one by one at different time periods, pellet down the cells and frozen for overnight at -70°C.Next day, the cells were thawed and sonicated for 30 s with the interval of 30 s and placed on ice for 30 s, this process was repeated 3 -4 times, then 1X SDS-PAGE loading dye was added.The protein samples were boiled at 100°C for 10 min, and then placed at 4°C before loaded on SDS-PAGE gel analysis

Cloning of βC1 of ChLCB in P. pastoris vector
The resultant clone pSAβC1PET32a (+) and pPIC3.5Kwere Andleeb et al. 8025 digested with EcoRI and NotI restriction enzymes.The digestion was run on 1% agarose gel and restricted fragment of expected size was eluted from the gel by DNA extraction kit (MBI Fermentas).Elution was confirmed by running 2 µl of gel extracted DNA on 1% agarose gel and purified fragment was ligated into P. pastoris expression vector at EcoRI and NotI.Ligation was transformed into 10 b heat shocked competent cells and colonies were selected on LB medium supplemented with ampicillin (100 mg/ml).The resultant recombinant vector was named pSAβC1PIC3.5K.
Transformation and screening of βC1 of ChLCB in P. pastoris GS115 strain Construct pSAβC1PIC3.5Kplasmid was isolated from E. coli strain by miniprep method, linearized with NotI restriction enzyme for integration in the genomic DNA of P. pastoris .Electrocompetent cells of P. pastoris GS115 strain were prepared by electrochemical method.The electroporation apparatus was adjusted to 2.0 kV.Electroporation cuvettes with a gap of 2 mm were used in this experiment.The GS115 electro competent cells (80 µl as removed from the -70°C freezer and thawed on ice) were transferred and pSAβC1PIC3.5K was linearized in the cuvettes, electric shock was given at 2.0 kV.Then immediately, 1 ml 1 M of sorbitol was added and the cuvettes were placed on shaker at 30°C for 2 h.After 2 h, the medium was centrifuged at 13200 rpm for 1 min to collect pellet.Pellet was dissolved in 100 µl 1 M sorbitol and spread on the yeast peptone dextrose (YPD) agar plates containing antibiotic geneticin up to final concentration of 0.25 mg/ml and these plates were transferred to incubator at 30°C until colonies appeared.Selection of transforments was carried out at different concentrations of geneticin antibiotic in YPD agar medium plates.These concentrations were 0.25, 0.5, 0.75, 1.0, 1.25 and 1.5 mg/ml, respectively.

Optimization of P. pastoris growth for SDS-PAGE analysis
P. pastoris transformants having pSAβC1PIC3.5Kwere picked from 0.75 mg/ml geneticin YPD agar media plates along with nontransformant GS115 as a control and cultured into YPD broth media without antibiotic at 30°C (OD600 = 1.0).Cells were harvested by centrifugation at 3000 rpm for 5 min at room temperature.Supernatant was discarded and cell pellet was resuspended into 25 ml minimal glycerol medium (MGM) and 0.02% 10 X dextrose in a 100 ml flask.Cultures were placed at 28 -30°C in a shaking incubator (150 -200 rpm) until growth reached log phase.Once the cells are in log phase, they can be induced for βC1expression. 1 ml culture was taken before each induction of 100% methanol to a final concentration of 30 µl in 25 ml MG medium.Induced culture was collected at different time intervals (24, 48, 72, 96 and 120 h), respectively.1 ml of expression culture was transferred into 1.5 microcentrifuge tubes.These samples were used to analyze expression levels and determine the optimal time from postinduction to harvest.Cells were centrifuged at 13,500 rpm at room temperature for 2 -3 min.For intracellular and secreted expression, both supernatant and pellet was stored at -70°C until ready for protein assay.

Preparation of βC1 of ChLCB protein sample in P. pastoris GS115 strain
The sample was prepared for SDS-PAGE, as cell pellets were thawed quickly and placed on ice.Pellet was dissolved in 1 ml distilled water and 100 µl breaking buffer.An equal volume of acid washed glass beads (size 0.5 mm) was added.It was vortex for 30 s, incubated on ice for 30 s (repeated for several times) then centrifuged at 13,500 rpm for 10 min.Clear supernatant was transferred to a fresh 1.5 microcentrifuge tube, 50 µl SDS-PAGE loading dye for SDS-PAGE analysis was added and boiled for 10 min at 100°C in a dry bath.10 -20 µl sample per well was loaded into SDS-PAGE gel, whereas, others used for xylanase assay and remaining were stored at -20°C for use in future.Electrophoresis was performed as described by using a discontinuous buffer system for the analysis and separation of proteins.Developer solution was added to enhance and visualized the bands during silver staining of SDS-PAGE of βC1 protein in P. pastoris.

Expression of βC1 through prokaryotic system
Cloning of βC1 gene into pET32a (+) vector (bacterial expression system) The PCR amplified 450 bp fragment using specific primers (Figure 2A) was cloned into T/A cloning vector pTZ57R/T.The resultant recombinant clone pSAβC1T/A was confirmed through digestion.Upon restriction with EcoRI and HindIII, it should produce 450 bps fragments (Figure 2B and C).The βC1gene was eluted, and cloned in the pET expression vector, which is confirmed through both restriction and PCR.Finally, pSAβC1pET32a(+) recombinant vector transformed into E. coli strain BL21 for bacterial expression.BL21 transformants having the target gene from pSAβC1pET32a (+) was confirmed through PCR analysis by using set of specific primers (Figure 2D).

SDS-PAGE analysis for βC1 protein induced by IPTG inducer
After transformation and confirmation of pSAβC1pET32a (+) and pET32a(+) in E. coli strain BL21, the samples were processed and analyzed on SDS-PAGE.The desired band could not be detected in the induced transformants having pSAβC1pET32a (+) with increase in time after 1 mM IPTG induction of 30, 60, 90 and 120 min, respectively.On the other hand, the other bands regarding to BL21 proteins were visible.We increased the level of IPTG and time of induction up to 7 h to expression βC1 in bacteria, but the experiment failed to detect protein on SDS PAGE analysis.However, βC1 was easily cloned into pDONR/Zeo gateway vector through gateway cloning, but it also failed (data not shown).

Isolation of βC1gene and construction of pSAβC1PIC3.5K recombinant vector
Both construct pSAβC1pET32a(+) and vector pPIC3.5kwas digested with EcoRI and NotI.Upon digestion of pSAβC1pET32(+), it produced 450 bp fragment from the vector backbone of pET 32a(+) vector, whereas, pPIC3.5kproduced a linear fragment of approximately 9000 bp.Restriction analysis indicated that EcoRI and NotI are unique multiple cloning restriction sites of pPIC3.5k,whereas, in pSAβC1PIC3.5K it is located on upstream and downstream of gene.The elution of the target fragments of approximately 450 and 9000 bp, respectively, was carried out through DNA extraction kit (Fermentas) and eluted bands were analyzed through running on 1% agarose gel electrophoresis.Eluted products were ligated at 16°C.βC1 gene from pSAβC1p ET32 was cloned at the same sites of EcoRI and NotI in pPIC3.5K.The resultant vector was named pSAβC1 PIC3.5K.

Confirmation of clone pSAβC1PIC3.5K in E. coli TOP10F´strain
Ligation product was transformed into E. coli TOP10F´ strain by heat shock method.The recombinant clone pSAβC1PIC3.5K was confirmed through digestion analysis.
Upon digestion with EcoRI and NotI, recombinant clone pSAβC1pPIC3.5Kproduced 450 bp fragment along with vector backbone of pPIC3.5K of 9000 bp, mobilized into E.coli TOP10F´ strain for its mass production before transforming in P. pastoris GS115 strain.

Expression of βC1 gene in P. pastoris (yeast expression system) GS115 strain: Transformation and selection of recombinant Pichia clones carrying foreign gene through PCR
pSAβC1pPIC3.5Kplasmid was isolated from TOP10F´ E. coli strain and linearized with NotI restriction enzyme.The linearized fragment was treated with phenol: chloroform for purification and transformed into P. pastoris strain GS115 through electroporation.Concentration of geneticin was optimized for the selection of transformants from 30, 50, 70, 90, 110 and 130µg/ml.Suitable concentration of geneticin antibiotic for selection of transformants was found to be 0.25 mg/ml.The transformants were grown on YPD agar media plates containing 0.75 mg/ml geneticin antibiotic.

Confirmation of the transformants in P. pastoris GS115 strain
The integration of linearized fragment pSAβC1pPIC3.5Khaving βC1 gene in the genome of P. pastoris was confirmed through PCR amplification by using βC1specific primers (Figure 3).The amplified 450 bp fragment from the transformants of P. pastoris having pSAβC1PIC3.5K was confirmed through 1% agarose gel electrophoresis.

Confirmation of βC1 gene expression
The expression of targeted gene in P. pastoris GS115 strain transformants having recombinant vector pSAβC1p PIC3.5K was confirmed from four selective GS115 cultures.The expression was induced by the of 100% methanol in minimal glycerol broth media without geneticin.The strains transformed by parent plasmid pPIC3.5Kwere taken as control.The samples were collected at different times of induction (after 24, 48, 72 and 96 h), respectively.These samples were processed and analyzed on SDS-PAGE.The intensity of desirable protein approximately 26 kDa was increased with increase in time after 100% methanol up to a final concentration of 0.5% induction.The band of the same molecular mass could not be detected in the induced recombinant strain transformed pPIC3.5Kvector and the results showed that the recombinant protein was successfully expressed (Figure 5).The protein bands were clearly visible only in the case of the sample that was incubated for 72 h and no clear bands were observed after 24, 48 and 96 h of incubation.Studies conducted further to scale-up the expression level with increased induction period and methanol concentration revealed 72 h of post-induction incubation period with 1.5% methanol concentration for large amount of βC1 protein expression.However, the level of expression could not be further boosted above this scale with either increased methanol concentration or increased duration of incubation.

Bacterial expression of βC1
Betasatellite gene βC1 was successfully cloned into bacterial expression vector pET32a (+) and transformed into BL21 E. coli strain for expression, but βC1 was not expressed in prokaryotic system after several trial experiments.Similar results were obtained when we cloned βC1 through gateway cloning vector.We observed that when we cloned βC1 into pDONR/Zeo vector after confirmation through PCR and restriction analysis, it produced same fragment of βC1, but it showed nucleotide mutations through sequence analysis after several trials of cloning.Figure 4A describes the alignment of different clones of gateway vectors having βC1 through clustal W (1.8) aligner program at http://www.justbio.com/tools.php.These selected recom-binant clones were translated through translator program at http://www.justbio.com/tools.phpand also aligned through above clustal W (1.8) program.The proteins alignment demonstrates the changes in the order of amino acids as indicated in Figure 4B.Both nucleotide and amino acid sequences of recombinant gateway vectors having βC1 shows homology with reported sequence from Genebank nucleotide βC1 sequence database accession no.AJ316032, so we can say that βC1 is a lethal protein for bacterial system and could not be expressed.Later we cloned βC1 into Pichia vectors for expression in yeast because in P. pastoris, we can express any kind of protein.

P. pastoris expression of βC1
The P. pastoris system used in the present study is able to utilize methanol as its sole carbon source and has been widely used as a host for the expression of heterologous proteins.In this study, the βC1 gene of ChLCB was inserted towards the downstream of AOX1 promoter of the expression vector pPIC3.5Kand the chimeric construct was integrated into the host genome through homologous recombination.The recombinant yeast can also perform many eukaryotic post-translational modifications in the target protein, such as glycosylation, disulfide bond formation and proteolytic processing.Previous studies demonstrated that P. pastoris was capable of expressing glycoproteins processed in a manner similar to virus infected insect cells (Trimble et al., 1991).As the target gene is integrated within the genome, it is difficult to lose the target gene when the recombinant yeast is cultured.Therefore, P. pastoris has been used successfully to express a wide range of heterologous proteins (Andleeb et al., 2008;Romanos, 1995;Hagenson et al., 1989;Sreekrishna et al., 1997).Heterologous expression in P. pastoris can be either intracellular or secreted.Secretion requires the presence of a signal sequence on the expressed protein to target it to the secretary pathway.Although all four positive colonies were selected for induction, only two colonies showed expression of the target protein and the remaining two did not show detectable levels of the target protein in the gel for which reasons are not known.
The actual size of the gene 450 bp has been confirmed through PCR analysis (Figure 3) and the expected size of protein should be 16.5 kDa but SDS-PAGE analysis showed approximately 26 KDa molecular weight of a specific protein, which is expressed in P. pastoris, whereas, the protein sample transferred from Pichia transformed with pPIC3.5Knegative control did not develop any signal on the SDS membrane (Figure 5).The protein expression in P. pastoris was secreted and not intracellular.There may have been glycosylation which would make the protein larger and possibly produce multiple bands through multiple integration of pPIC3.5krecombinant vector with genomic DNA of Pichia.In order to increase the expression level, the expression conditions were optimized.The protein bands were clearly visible only in case of the samples that were incubated for 72 h and no clear bands were observed before or after 72 h of incubation.This could be due to the critical incubation Figure 5. Expression taken out up to 96 h.Lanes 1 -8, Pichia vector pPIC3.5Khaving chili βC1; and lanes 9 and 10, GS115 having pPic3.5Kvector.Lanes 1 and 5, after 96 h; lanes 2 and 6, after 72 h; lanes 3 and 7, after 48 h; lanes 4 and 8, after 24 h; PL (protein ladder), SM0661.period, that is, 72 h required for biomass accumulation.Very faint but multiple bands were observed following 72 h and no bands were visible after 120 h of incubation indicating that host-specific proteases may be acting on the protein following prolonged incubation.The observed low level of βC1expression may be partly attributed to the low copy number of the genes integrated within the yeast genome and partly to the nature of the protein.The most important parameter for efficient expression in Pichia was found to be adequate aeration during methanol induction and hence the culture volume within the flask was kept as low as 20% of the total flask volume.It was also necessary to maintain the incubation temperature at 28°C with rotation of 250 -300 rpm, above this temperature, yeast does not survive.

Conclusion
In conclusion, the pSAβC1PIC3.5Krecombinant protein has been expressed for the first time in both bacterial and yeast expression systems in our laboratory of Molecular Virology/Gene silencing (NIBGE, Pakistan).Our studies indicate that P. pastoris can be an excellent expression system for recombinant proteins.The P. pastoris expression system offers economy, ease of manipulation, the ability to perform complex post-translational modifications and high expression levels.Its use as an expression system will become increasingly more efficient and userfriendly.On the other hand it was also observed through sequence analysis that βC1 gene of ChLCB had mutations in nucleotide sequence when expressed in the bacterial system and could not be able to produce required protein and in modified form.It was suggested that may be it is due to its toxic effect in the system.The pSAβC1PIC3.5K recombinant vector constructed in the present study is suitable for secretary expression of the target protein but the expression of target protein was low and could not be purified.So, in future, we can modify the system for overproduction of pSAβC1PIC3.5K, to purify and use it in binding studies.

Figure 1 .
Figure 1.Chili plants showing the characteristic symptoms of virus infection like leaf curling, yellowing, vein thickening, distortion of leaves and stunting.

Figure 2 .
Figure2. Cloning of βC1 gene into pET32a(+) vector (bacterial expression system).The PCR amplified 450 bp fragment using specific primers (A) was cloned into T/A cloning vector pTZ57R/T.The resultant recombinant clone pSAβC1T/A, was confirmed through digestion (B).After digestion with EcoRI and HindIII, the βC1gene was cloned in the pET expression vector and verified with restriction enzymes (C).Finally pSAβC1pET32a(+) recombinant clone transformed into E. coli strain BL21 for bacterial expression.BL21 transformants having the target gene from pSAβC1pET32a(+) was confirmed through PCR analysis by using set of specific primers (D).
Fasteris SA, Plan-les-Ouates, confirmation of the exact size of the βC1 in pDONR/ Zeo through restriction and PCR analysis, the sequence of some of recombinant clones were analyzed through Fasteris SA, Plan-les-Quates, Switzerland.The alignment of different clones of gateway vectors having βC1 through clustal www.justbio.com/tools.php and by using DNAstar program (Figure IV.A).These selected recombinant clones were translated through translator program at http://www.justbio.com/tools.php and also aligned through above clustal W (1.8) program.The proteins alignment demonstrates the changes in the order of amino acids as indicated in (Figure IV.B).

Figure 4 :
Figure 4: Alignment of nucleotide and amino acid sequence analysis of βC1 in gateway vectors.(A)Indicates the nucleotide sequence homology of recombinant clones of gateway vector having βC1 gene through Clustal W (1.8) program with reported βC1 of NIB16.With solid deep red shade exactly matched with NIB16 and solid dark green color indicated mismatched with NIB16.(B)Shows the amino acid homology of recombinant clones of gateway vector having βC1 gene through Clustal W (1.8) program with reported βC1.Colorless amino acid indicates the differences between the sequences.