Indian Journal of Biotechnology

Vol 6, July 2007, pp 329-335

Expression of 3AB protein of foot and mouth disease virus in Pichia pastoris

M Latha Priyadharshini1, V Balamurugan, K Prabhudas, V V S Suryanarayana and G R Reddy*

Molecular Virology Laboratory, Indian Veterinary Research Institute, Hebbal, Bangalore 560 024, India 1Department of Microbiology, Madras Veterinary College, Chennai 600 007, India.

Received 19 August 2005; revised 5 July 2006; accepted 15 Ocotber 2006

3AB non structural protein (nsp) was used to diagnose the vaccinated animals from those infected with foot and mouth

disease virus (FMDV). In order to express the gene encoding 3AB protein of FMDV type A22 in Pichia pestoris, the gene

was amplified and cloned into the yeast transfer vector (pPIC-9K) at EcoRI site. The cloned gene was further characterized

by colony PCR, restriction enzyme digestion and sequence analysis. The recombinant plasmid was transferred into GS115

strain of P. pastoris cells by electroporation. The His+ Pichia transformants were analyzed for the presence of the insert in

the yeast genome by PCR. PCR positive clones were grown and expression was induced with 0.5% methanol. The expressed

gene products were then characterized by SDS-PAGE and Western blot analysis. This is the first report on the production of

FMDV non structural proteins in yeast. The expressed protein will be of diagnostic importance.

Keywords: 3AB protein, expression, FMDV, Pichia pastoris

IPC Code: Int. Cl.8 C12N 15/09, 15/11

Introduction

Foot and mouth disease virus (FMDV) causes a

devastating disease in cattle, pigs and sheep. The

disease can spread rapidly among susceptible

populations and has a great impact on the economy of

the affected countries. Where the disease is sporadic,

control of the disease is by slaughtering the affected

and in-contact animals, whereas in enzootic areas, the

control is by regular vaccination and restricted animal

movement. Animals recovered from the FMD can

become persistent carriers of the virus, in which case

the subjects carry the virus for several years1. The

carrier animals harboring the sub clinical infection

may be a source for new outbreaks. It is possible that

these carrier animals intermittently shed variants of

the virus, which differ antigenically from the original

strain2. Vaccination, thus, may be less effective in

these animals due to the variant viruses. Detection of

animals exposed to the disease in the livestock

population is very essential for effective FMD control

programmes. In fact, detection of replicating FMDV

in the vaccinated animals not manifesting clinical

signs is as relevant as the diagnosis of the acute

infection itself, since it acts as an indicator for vaccine

performance.

In endemic countries, like India, where regular

vaccination is carried out in the susceptible

population, majority of the susceptible animals are

sero-positive for structural proteins and it is not

possible to determine whether the animal is exposed

to the disease using whole virus as antigen. Since

non-structural proteins (nsps) are not present in

vaccine preparations3,4

, detection of antibodies to

these proteins is of diagnostic value whether the

animal/herd is exposed to the disease or not5.

Therefore, the present requirement is to develop a

serological test capable of differentiating vaccinated

from the FMDV infected animals. Such a test would

not only be useful to detect viral persistence but also

for serological surveys to be carried out for FMD

eradication. The test so developed should be highly

specific and sensitive, simple, innocuous and

inexpensive.

Nsps 2C, 3AB, 3ABC expressed in different

expression systems have been used in the ELISA and

other serological tests to detect the antibodies in

infected animals 6. The recent reports have suggested

that antibodies to 2C/3ABC/3AB could be the best

serological indicator of infection with FMDV.

However, these nsps produced in Escherichia coli

showed poor reactivity with sera of infected animals

probably due to poor solubility of the protein

expressed in bacteria7. Though the proteins produced

in insect cells are soluble, maintenance of the insect

______________________

*Author for correspondence:

Tel: 91-80-23418078; Fax: 91-80-23412509

E-mail: grreddy@rediffmail.com

INDIAN J BIOTECHNOL, JULY 2007

330

cells is laborious and costly. Yeast expression system,

on the other hand, has several advantages over

bacterial and insect cell systems, i.e. good over

expression level, secretion of protein into the medium,

ease of maintenance of the cells and scaling up

production8. The present study reports the expression

of 3AB in the yeast, Pichia pastoris.

Materials and Methods Virus

FMDV serotype A (Ind 17/77) vaccine strain was

grown in BHK-21 cl 13 monolayers. The seed virus

was obtained from the FMD vaccine production

laboratory of the Institute.

Sera Samples and Conjugate

Rabbit hyper immune serum against recombinant

3AB of FMDV expressed in E. coli and rabbit anti-

bovine IgG-HRPO conjugate, available in the

laboratory, were used. Goat anti-rabbit IgG-HRPO

conjugate was procured from Bangalore Genei

Bangalore, India.

Host Strain and Plasmid Vector

P. pastoris host strain, used in the study, was the

histidine requiring auxotroph GS115 (His4) pPIC-9K,

whereas yeast transfer vector contained AOX1

promoter and transcription termination sequences

along with MCS for insertion of the foreign genes of

interest and wild type copy of the histidinol

dehydrogenase (His4) gene for selection of P.

pastoris transformants. Both GS115 (His4) host strain

as well as plasmid pPIC-9K were procured from

Invitrogen, USA.

Amplification and Cloning of 3AB

Total RNA was extracted from infected BHK-21

cell culture supernatant using Trizol reagent

(Invitrogen, USA). The cDNA copy was synthesized

using the purified RNA as a template and 3dt

(5′ dTTTTTTAAAGAAAAGGAAG-OH3′) as a

primer with M-MLV (Molony Murine Leukemia

Virus) reverse transcriptase as per standard procedure.

The 3AB sequence was amplified from the cDNA

using 3Aln (5′d-GGT GAT TGA CCG GGT TGA G-

OH 3′) and VPgR (5′d- GAC TAT CGA ATT CTT

AGC TTT CTC -OH 3′) as upstream and downstream

primers, respectively. All the primers were designed

based on the published FMDV A12 sequence data9. A

50 µL reaction mix containing 1.5 mM MgCl2, 100

µM each of dNTPs, 25 mM Tris-HCl, 50 mM KCl, 20

pmol of each primer and one unit of Taq DNA

polymerase was amplified in the DNA Thermal

Cycler 9600 (Perkin-Elmer Cetus, USA) with initial

denaturation at 94ºC for 3 min, followed by 35 cycles

of 94ºC for 1 min, 60ºC for 1 min and 72º C for 1 min

with a final extension at 72ºC for 10 min.

The amplified PCR product corresponding to 3AB

(672 bp) gene was purified using wizard PCR

prep DNA purification system (Promega, USA),

digested with EcoRI and the digested product

(589 bp) was cloned into the pPIC-9K at

EcoRI site. The recombinant colonies grown on

kanamycin plates were initially screened

by colony PCR, using vector specific 5′AOXI

(5′dGACTGGTTCCAATTGACAAGC-OH3′) and

insert specific VPgR (3’end of the insert) primer to

check orientation of the insert, followed by insert

release from the recombinant plasmid DNA. The

recombinant plasmid with correct orientation of the

insert (pPICA22-3AB) was used for further study.

Sequencing of 3AB Gene

The 3AB insert in plasmid (pPIC-9K) was

sequenced with 3'AOX1 (5'dGCAAATGGCATT-

CTGA-OH3') and 5’AOX1 primers using the ABI

377 automated DNA sequencer (ABI Inc., USA) to

confirm the frame and specificity of the cloned

fragment. The sequence reaction was carried out

using 1 µg of plasmid DNA and 2 pmol of the primer.

Sequence data obtained was analyzed with the help of

Omiga 1.13 DNA analysis software. Both nucleotide

and derived amino acid sequences were compared

with A12 sequence.

Expression of Cloned 3AB in P. pastoris

The pPICA22-3AB plasmid was linearized with

SalI and transferred into GS115 strain of P. pastoris

by electroporation for 10 msec with field strength of

7500 V/cm using Gene Pulsar II (Bio-Rad, USA).

Transformants harbouring the plasmid-borne His4

marker were selected on minimal plates lacking

histidine. Yeast chromosomal DNA was extracted

from the transformants (Mut+) by spheroplasting with

zymolyase, followed by phenol:chloroform

extraction.

Presence of the insert in the yeast genome was

confirmed by PCR amplification using 5’ AOX1 and

3’AOX1, vector specific primers. Induction of the

protein expression was carried out by standard

procedures8. Single colony of the His

+Mut

+ positive

clone and the vector transformant of Pichia was

inoculated in 25 mL buffered glycerol-complex

PRIYADHARSHINI et al.: EXPRESSION OF FMDV 3AB PROTEIN GENE IN PICHIA PASTORIS

331

medium (BMGY), kept in two 250 mL flasks, and

incubated at 30°C in a shaking incubator (250-300

rpm) to reach an A600 of 6. The cells were harvested

and suspended in buffered methanol-complex medium

(BMMY) to an A600 of 1.0 (about 100-200 mL

medium) and incubated at 30°C, and methanol was

added to a final concentration of 0.5% at every 24 h

interval up to 96 h. After 96 h incubation, entire

culture supernatant was harvested and the secreted

proteins were precipitated with 50% ammonium

sulfate, and dialyzed against phosphate buffered

saline (PBS, pH 7.4)). The protein content was

estimated by Bradford method11

with bovine serum

albumin (BSA) as the standard. Partially purified

proteins, from the recombinant Pichia clone along

with control pPIC-9K were separated on 15%

polyacrylamide gels under denaturing conditions

(SDS-PAGE) as per the method of Laemmli12

.

A similar duplicate gel was blotted onto a PVDF

membrane for immuno detection. The recombinant

protein was detected by treating with anti-3AB rabbit

serum (at 1:800 dilution), followed by an anti-rabbit

antibody HRPO conjugate (1:1000) and

orthodianisidine dihydrochloride (ODD) as

substrate11

.

Results Cloning of 3AB in Yeast Transfer Vector

The 3AB-coding sequence was amplified by RT-

PCR using RNA extracted from the virus, which

resulted in the amplification of specific PCR product

of 670 bp (Fig. 1, lane 2). The amplified product was

purified, digested with EcoRI and ligated with EcoRI

digested pPIC-9K vector. Presence of the insert

(containing 3B and truncated 3A) in the recombinant

plasmids was confirmed by EcoRI digestion. Release

of 0.6 kb fragment confirmed the presence of the

insert. Orientation of the insert in the plasmid was

confirmed by PCR with 5’AOX1 (vector specific) and

VPgR (virus specific) primers. Amplification of 1 kb

product (Fig. 2, lane 1) showed that the insert is in 5’-

3’ orientation whereas amplification of 0.4 kb product

indicated the 3’-5’ orientation (Fig. 2, lanes 2 & 3). Sequencing of 3AB Clone

Nucleotide and the amino acid sequence of cloned

3AB gene of serotype A (Indian vaccine strain) is

shown in Fig. 3. The cloned 3AB gene is of 672

nucleotides and codes for 224 amino acids. After

digestion with EcoRI, the PCR fragment (589 bp) was

cloned into pPIC-9K, a yeast transfer vector, and

Fig. 1—Agarose gel electrophoresis of amplified PCR products:

lane 1, Negative control (without cDNA); lane 2, PCR product

(3AB) amplified from cDNA; & lane M, Standard DNA

molecular weight marker (100bp ladder, Invitrogen, USA).

Fig. 2—Agarose gel electrophoresis of PCR products amplified

from recombinant plasmids with 3AB inserts: lane M, Standard

DNA molecular weight marker (100bp ladder Invitrogen, USA);

lane 1, Amplified PCR products from the plasmid with 3AB insert

in correct orientation; lanes 2 & 3, Amplified PCR products from

the plasmids with 3AB insert in reverse orientation; & lane 4,

pPIC-9K vector control.

INDIAN J BIOTECHNOL, JULY 2007

332

frame and orientation of the insert was confirmed by

sequencing. The insert was in frame with vector

coded ATG codon. The sequence of the cloned insert

and the deduced amino acid sequence translated in +3

frame as shown in Fig. 3. To examine the relation

with reported sequence of the serotype A (A12 strain),

nucleotide and the amino acid sequences of both the

strains were aligned (Fig. 4), which indicated a

homology of 90% in the nucleotide sequence and

96% in amino acid sequence, confirming that the

sequence cloned was of 3AB gene. Variation in the

amino acid sequence was more pronounced in 3A

compared to the 3B protein.

Transformation of pPICA22-3AB into Pichia pastoris

In order to express the 3AB gene, recombinant

plasmid (pPICA22-3AB) was linearized with SalI and

was transferred into GS115 strain of Pichia by

electroporation with linearized pPIC-9K vector as

control. The recombinant Pichia clones were screened

for confirming the presence of 3AB insert in the yeast

genome by PCR using vector specific primers.

Histidine positive clones with at least one integrated

copy of the expression cassette can be easily

distinguished from un-transformant colonies by

comparing the size of PCR products amplified in PCR

colony using the 5’AOX1 and 3’AOX1 primers. The

Fig. 3—Nucleotide sequence and deduced amino acid sequence of 3AB of FMDV serotype A ( Indian strain).

PRIYADHARSHINI et al.: EXPRESSION OF FMDV 3AB PROTEIN GENE IN PICHIA PASTORIS

333

chromosomal DNAs from the individual clones were

prepared and subjected to PCR. Amplification of

specific 1.1 kbp product (corresponding to 612 bp

insert and rest is from the vector) could be seen in the

gel (Fig. 5, lanes 1 & 2). Of the five colonies screened

by PCR for integration, two were found to carry the

vector with 3AB insert, which were used for protein

expression.

Expression of 3AB in Pichia pastoris

The PCR positive Pichia clones were grown

separately and the expression was induced with 0.5%

methanol. Since the inserted gene is present

downstream to the secretary signal sequences, the

expressed gene product was expected to be secreted

out in the medium. Proteins expressed by recombinant

clones carrying integrated DNA with and without

insert in the culture supernatants were analyzed by

SDS-PAGE, followed by Western blot analysis. A

protein band of 26 kDa was seen in the gel (Fig. 6,

lane 3) corresponding to 3AB protein. Protein bands

separated in the gel were transferred on to PVDF

membrane and detected with antiserum specific to 3AB

(Fig. 7). An intensive color reaction was observed with

the protein band size corresponding to 26 kDa from

recombinant clone (Fig. 7, lane 1) which confirms the

expression of 3AB protein by P. pastoris.

Discussion

In India, diagnosis based on the detection of

antibodies to viral nsps is important to assess the

prevalence of endemic disease. The antibodies against

Fig. 4—Alignment of amino acid sequences of 3AB of FMDV Sero type A strains.

INDIAN J BIOTECHNOL, JULY 2007

334

virus capsid show the sero-conversion in the

vaccinated animals, while the presence of antibodies

against nsps shows the disease prevalence situation

either due to previous out brakes or presence of the

residual live virus in case of vaccinated animals.

Diagnosis of infection at sub-clinical level is very

much relevant both in the control and eradication of

FMD in endemic areas and as a supportive measure to

the ‘stamping out’ policy in FMD-free areas. The poly

protein 3AB and 3ABC have been proposed as the

most antigenic of the FMDV nsps and it has been

argued that antibody specific for these proteins could

be the most useful marker of viral replication14,15

.

Hence, presence of antibodies against these nsps

certainly indicates viral multiplication in the animal.

Recently, the yeast has emerged as a powerful

heterologous expression system for the production of

high levels of functionally active recombinant

proteins8. To expedite such approaches that circumvent

several problems encountered in expressing the 3AB,

we expressed 3AB protein in yeast P. pastoris system.

The cloning of the 3AB sequence downstream to the

highly inducible AOX1 promoter and signal peptide

permitted high-level expression and secretion of the

recombinant protein. The translated product is in the

form of a fusion protein, which gets secreted out after

cleavage of the signal sequences. Using SalI linearized

pPICA22-3AB plasmid in Pichia, homologous

recombination between the transforming DNA and

region of homology within the yeast genome is

generally expected to occur within the His4 locus,

which leads to the generation of Mut+ recombinant of

GS115 cells2,10

. Electroporation method of transform-

ation in Pichia yielded 2×103 His+ transformants/µg of

DNA, which is less than reported in Saccharomyces

(103-10

4/µg of DNA)

17,18.

Fig. 5—Agarose gel electrophoresis of PCR amplified 3AB

inserts from Pichia clones: lane M, Standard molecular weight

marker (100bp ladder, Invitrogen, USA; lane 1 & 2, Amplified

PCR products from the chromosomal DNA of clones with 3AB

insert; lane 3, Amplified PCR products from the chromosomal

DNA of parent strain (GS115) showing the AOX 1 gene intact;

lane 4, Amplified PCR products from the chromosomal DNA of

clones with pPIC-9K; lane 5, Amplified PCR products from pPIC-

9K recombinant clone (positive control); lane 6, Amplified PCR

products from pPIC-9K vector; & lane M, Standard molecular

weight marker (100bp ladder, Bangalore Genei, Bangalore).

Fig. 6—SDS-PAGE Analysis of Expressed FMDV Protein: lane

1, Proteins from culture supernatant of yeast cells (GS115); lane

2, Protein from induced culture supernatant of yeast cells without

insert; lane 3, Protein from induced culture supernatant of yeast

cells with 3AB insert; & lane M, Standard protein molecular

weight marker (PMW-L, Bangalore, Genei, Bangalore).

Fig. 7—Western blot analysis of expressed FMDV protein; lane,

1: Culture supernatant of induced yeast cell culture carrying 3AB

insert; & lane 2, Culture supernatant of yeast cell carrying no

insert.

PRIYADHARSHINI et al.: EXPRESSION OF FMDV 3AB PROTEIN GENE IN PICHIA PASTORIS

335

The quantity of protein expressed by the

recombinant yeast clone was 200 mg/L culture

supernatant, which is in the range of 6.3 mg to 12g/L

culture depending on the nature of the protein as

reported by various groups18,19

. The calculated size of

the 3AB comes to 23 kDa, which is in agreement with

the protein size observed in SDS-PAGE. This indicates

that the product is from the cloned gene.

In conclusion, the results show the expression of

3AB gene of FMDV serotype A vaccine strain in P.

pastoris, which will be useful for development of

diagnostic test to differentiate the vaccinated from the

infected animals.

Acknowledgment

We wish to thank the Director, IVRI, Izatnagar and

Joint Director, IVRI, Bangalore for providing all the

facilities to carry out this work. We are indebted to

late Dr S M Lal, ex-Joint Director, IVRI, Bangalore

Campus, for his support and encouragement.

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