ORIGINAL PAPER

Translational fusion of chloroplast-expressed humanpapillomavirus type 16 L1 capsid protein enhances antigenaccumulation in transplastomic tobacco

Paolo Lenzi Æ Nunzia Scotti Æ Fiammetta Alagna Æ Maria L. Tornesello ÆAndrea Pompa Æ Alessandro Vitale Æ Angelo De Stradis Æ Luigi Monti ÆStefania Grillo Æ Franco M. Buonaguro Æ Pal Maliga Æ Teodoro Cardi

Received: 29 January 2008 / Accepted: 29 April 2008

� Springer Science+Business Media B.V. 2008

Abstract Human Papillomavirus (HPV) is the

causal agent of cervical cancer, one of the most

common causes of death for women. The major

capsid L1 protein self-assembles in Virus Like

Particles (VLPs), which are highly immunogenic

and suitable for vaccine production. In this study, a

plastid transformation approach was assessed in order

to produce a plant-based HPV-16 L1 vaccine.

Transplastomic plants were obtained after transfor-

mation with vectors carrying a chimeric gene

encoding the L1 protein either as the native viral

(L1v gene) or a synthetic sequence optimized for

expression in plant plastids (L1pt gene) under control

of plastid expression signals. The L1 mRNA was

detected in plastids and the L1 antigen accumulated

up to 1.5% total leaf proteins only when vectors

included the 50-UTR and a short N-terminal coding

segment (Downstream Box) of a plastid gene. The

half-life of the engineered L1 protein, determined by

pulse-chase experiments, is at least 8 h. Formation of

immunogenic VLPs in chloroplasts was confirmed by

capture ELISA assay using antibodies recognizing

conformational epitopes and by electron microscopy.

Keywords Plastid transformation � HPV16 �L1 � Plant vaccines � Tobacco � Nicotiana tabacum

Introduction

Human Papillomaviruses (HPVs) are a group of more

than 100 types of double-stranded and nonenveloped

DNA viruses associated with both benign and

malignant epithelial lesions (de Villiers et al. 2004).

Among these, approximately 40 HPV-types infect the

anogenital mucosa and eight ‘‘high-risk’’ types (16,

18, 31, 33, 35, 45, 52, 58) account for 95% of

cervical cancer worldwide (Munoz et al. 2003).

HPV16, in particular, is found in more than 50% of

Contribution No. 129 from CNR-IGV, Portici.

P. Lenzi � N. Scotti � F. Alagna � L. Monti �S. Grillo � T. Cardi (&)

CNR-IGV, Institute of Plant Genetics-Research Division

Portici, via Universita 133, 80055 Portici, Italy

e-mail: cardi@unina.it

P. Lenzi � P. Maliga

Waksman Institute of Microbiology, Rutgers, The State

University of New Jersey, 190 Frelinghuysen Road,

Piscataway, NJ 08854-8020, USA

M. L. Tornesello � F. M. Buonaguro

Viral Oncogenesis and Immunotherapy, Department

of Experimental Oncology, Istituto Nazionale Tumori

‘‘Fondazione Senatore G. Pascale’’, Via M. Semmola n.1,

80131 Napoli, Italy

A. Pompa � A. Vitale

CNR-IBBA, Istituto di Biologia e Biotecnologia Agraria,

via Bassini 15, 20133 Milano, Italy

A. De Stradis

CNR-IVV, Institute of Plant Virology, Via Amendola

165/A, 70126 Bari, Italy

123

Transgenic Res

DOI 10.1007/s11248-008-9186-3

cancer cases. Cervical cancer is the most common

malignancy of women in developing countries, with

about 500,000 new cases worldwide each year

(Clifford et al. 2003). For this reason, the develop-

ment of a preventive vaccine is nowadays a high-

priority need in human medicine. L1, the major

capsid protein, is able to self-assemble and to form

Virus Like Particles (VLP) that mimic the infection

with virions and induce the production of virus-

neutralizing antibodies. Results from clinical studies

in human subjects indicate that HPV VLPs are safe,

well tolerated and highly effective (Frazer 2006;

Schiller and Lowy 2006; Stanley 2006; Leggatt and

Frazer 2007). Hence, despite some ‘‘remaining ques-

tions’’, concerning mainly the duration of protection,

the cross-type protection, and the protection in men,

L1-based prophylactic vaccines, produced either in

insect or yeast cells, have reached the commercial-

ization phase (Frazer 2006; Schiller and Lowy 2006).

Plants represent an alternative production platform

for the production of biopharmaceuticals, therapeutic

proteins, and recombinant subunit vaccines to the

conventional expression systems, such as yeast,

bacteria, insect and mammalian cell cultures. The

advantages of ‘‘molecular farming’’ in plants over

conventional production systems are low cost, ease of

scalability, possibility to deliver unprocessed or

partially processed material, and low health risks,

because plant cells are not hosts for human pathogens

(Daniell et al. 2001; Ma et al. 2003; Fischer et al.

2004; Ma et al. 2005). At present, three plant-derived

proteins reached commercialization and many others

are under-going clinical trials (http://www.molecular

farming.com). Plant-based production of a HPV-16

L1-vaccine has also been attempted. Incorporation of

the HPV L1 coat protein gene in the plant nucleus,

with one exception (Maclean et al. 2007), yielded

only very low levels of protein expression (Biemelt

et al. 2003; Varsani et al. 2003; Liu et al. 2005).

We report here an alternative approach to express

the HPV16 L1 capsid protein from genes incorpo-

rated into the plastid genome. This technology has

been employed to achieve high levels of protein

expression in chloroplasts, due to high gene copy

number and absence of epigenetic effects (gene

silencing, position effects) (Maliga 2002; Daniell

2006; Bock 2007; Verma and Daniell 2007). Obtain-

ing high protein levels was dependent on mRNA

levels, the translatability of the mRNAs and protein

degradation (Maliga 2002). Additional potential

advantages of the plastid-expression system rely on

the feasibility of expressing multiple proteins from

polycistronic mRNAs and transgene containment due

to maternal inheritance of plastids, thus avoiding

transgene spreading through pollen, in most crops

(Bock 2007; Murphy 2007; Verma and Daniell 2007).

In this study, the HPV-16 L1 protein, derived by

either a viral or a codon-optimized sequence, was

expressed under the control of different 50-UTRs with

or without a Downstream Sequence (Maliga 2002).

N-terminal translational fusion of the L1 protein with

the plastid photosynthetic proteins was necessary to

obtain detectable levels of protein accumulation.

ELISA assays with conformation-sensitive antibodies

indicate that the L1 protein assembled in immuno-

genic higher-order structures, such as VLPs and

capsomeres. Relatively high yield of L1 protein and

its correct assembly into immunogenic VLPs make

plastid expression a promising approach for devel-

oping a low cost HPV vaccine.

Materials and methods

Plasmid construction

The HPV-16 African-1 L1v sequence (GenBank

Accession No. AF472508) was obtained from a

cervical carcinoma DNA sample of an Ugandan

patient (Buonaguro et al. 2000) by PCR amplification

with forward 50-GAagatctATGTCTCTTTGGCTGC

CTAGTGA-30 and reverse 50-GGGaagcttCAATACT

TACAGCTTACG-30 primers (restriction sites are in

lowercase). The PCR product was first digested with

HindIII, followed by Klenow-blunting and digestion

with BglII to originate compatible ends for inserting

into SmaI/BamHI sites of pGEX2T (GE Healthcare).

In order to construct pPL20, pPL22 and pPL24

vectors, the gene fused to the Glutathione S-Trans-

ferase (GST) in pGEX-2T plasmid (GST-L1v

sequence) was PCR-amplified adding a NheI site at

the 50 end and an XbaI site at the 30 end. Plasmid

pPL20 is a pHK30 (Kuroda and Maliga 2001a) vector

derivative in which the NheI–XbaI fragment encoding

neo was replaced with a NheI–XbaI fragment encod-

ing a GST-L1v fusion protein. Plasmid pPL22 is a

pHK35 (Maliga et al. 2001) derivative in which the

GST-L1v sequence replaces neo as a NheI–XbaI

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123

fragment. Plasmid pPL24 is a pMHB10 derivative

(Lutz et al. 2006) in which the GST-L1v gene

replaces the bar gene. In this vector, the transgene

is regulated by a PrrnLpsbA cassette and it is

translationally fused upstream of the aadA marker

gene in a dicistronic construct; the marker gene is

flanked by two lox loci. Plasmid pNS31 is a pHK35

derivative, in which a His-L1v replaces the neo gene.

To construct vectors containing the plastid-opti-

mized L1 (L1pt), the native protein sequence, without

the last 22 amino acids encoding the Nuclear Local-

ization Signal (Zhou et al. 1991), was back translated

using the Backtranslation tool in Entelechon web site (

http://www.entelechon.com). Plasmids pPL61 and

pPL66 are pHK34 (Kuroda and Maliga 2001a) deriv-

atives, in which the L1pt gene replaces the neo coding

sequence. In pPL61, the 6xHis tag is cloned on the L1

N-terminus, while in pPL66 it is on the C-terminus.

Plasmids pPL62, pPL63 and pPL70 are pHK35 (Ma-

liga et al. 2001) derivatives with L1pt (L1pt-gfp in

pPL70) cloned in place of the neo sequence. For

pPL63, a truncation was performed obtaining a DC205

aa version. For pPL70 vector, the following oligo

50-TTATgagctccctaggAGCTGGTGTTGCCTCCGGA

GCCGTAGGTTCTGGAGccatggctagcATTA-30 and

its complementary 50-AATActcgagggatccTCGACC

ACAACGGAGGCCTCGGCATCCAAGACCTCggt

accgatcgTAAT-30 (restriction sites are in lowercase),

encoding a 12 amino acids linker, were annealed and

cloned between L1pt and gfp gene in a pPL62 deriva-

tive. Plasmid pPL69 is a pHK40 (Kuroda and Maliga

2001b) derivative, with L1pt in place of the neo gene. In

all vectors, promoters and other regulatory sequences

were fused to L1pt gene via an engineered NheI site,

using the compatible SpeI site. Details about cloning

strategy are available upon request.

Plastid transformation

Plants of Nicotiana tabacum cv. Petit Havana were

grown in sterile conditions on MS medium (Murash-

ige and Skoog 1962). Leaves from 3- to 8-week-old

plants were used for bombardments. DNA for plastid

transformation was prepared using the Qiagen Plas-

mid Maxi Kit (Qiagen). DNA was introduced into

leaf chloroplast on the surface of tungsten (1 lm) or

gold particles (0.6 lm), using the DuPont

PDS1000He Biolistic Gun. Transplastomic shoots

were produced with vectors reported in Fig. 1 and

with the empty vector pPRV111A (Zoubenko et al.

1994), after selection on RMOP medium containing

500 mg/l spectinomycin dihydrochloride. The trans-

genic plants were grown on MS medium (Murashige

and Skoog 1962) containing 3% (w/v) sucrose and

0.8% (w/v) agar in sterile culture conditions. In some

cases, 500 mg/l spectinomycin was also added to

propagation medium. Integration of recombinant

DNA and homoplasmy of the transplastomic plants

were confirmed by DNA gel-blot analysis. Double

strand DNA probe was prepared by random priming32P labeling using the Ready-To-Go DNA Labeling

Beads (GE Healthcare). The probe was the ApaI–

BamHI ptDNA sequence in rrn16-rps12 flanking

regions. Hybridization to the probe was carried out in

Rapid Hybridization Buffer (GE Healthcare) over-

night at 60�C.

RNA gel blot analysis

Total cellular RNA was isolated from leaves of plants

grown in sterile conditions (Stiekema et al. 1988).

Total RNA of 5 lg were loaded per lane and

electrophoresed on 1% agarose-formaldehyde gel

and transferred to Hybond N membrane (GE Health-

care) using the PosiBlot Transfer Apparatus

(Stratagene). Probes were prepared by random-prim-

ing 32P-labeling (see above). The probe used was a

gel-purified GST-L1 NheI–XbaI fragment amplified

from pPL20 for L1v plants and a L1 NdeI–XbaI

fragment excised from pPL53 plasmid for L1pt plants.

SDS-PAGE and immunoblot analysis

Leaves for protein extraction were collected from

plants grown in sterile conditions. To extract soluble

proteins, 200 mg leaves were homogenized in 1 ml

buffer containing 150 mM Tris–HCl (pH 7.5),

150 mM NaCl, 1.5 mM EDTA, 2% b-mercaptoeth-

anol, 2% SDS and 2 mM PMSF. The extract was

heated for 5 min at 95�C.

Proteins were separated by SDS-PAGE (12.5%

acrylamide gel) and transferred onto nitrocellulose

membranes using a semi-dry transfer apparatus

(Bio-Rad). Blocked membranes were incubated with

1:20,000 diluted anti-L1 monoclonal antibody

(Camvir 1, Millipore) (McLean et al. 1990). Chem-

ioluminescence reagent (GE Healthcare) was used for

immunoblot detection on X-ray film (Kodak).

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123

Pulse-chase experiments

Protoplasts were prepared from small (4–7 cm) leaves

of in vitro-grown plants, as described by Pedrazzini

et al. (1994). Radioactive labeling was performed by

incubation at 25�C in the dark in K3 medium

(Gamborg’s B5 basal medium with minimal organics

(Sigma), supplemented with 750 mg/l CaC12 � 2H2O,

250 mg/l NH3NO4, 136.2 g/l sucrose, 250 mg/l

xylose, 1 mg/l 6-benzylaminopurine, and 1 mg/l

a-naphthalenacetic acid, pH 5.5) supplemented with

150 lg/ml of BSA and 100 lCi/ml of PRO-MIX

(a mixture of 35S-methionine and cysteine; GE Health-

care). Chase was performed by adding unlabeled

methionine and cysteine up to a final concentration of

10 and 5 mM, respectively, from a 109 concentrated

stock. At the desired time points, 3 volumes of ice-cold

W5 medium (154 lM NaCl, 5 lM KCl, 125 lM

CaCl2 � 2H2O, 5 lM glucose) were added, and sam-

ples were centrifuged for 5 min at 50g, 4�C. The

supernatant, containing secreted proteins, was

removed, leaving 100 ll to cover the protoplasts. The

supernatant and protoplasts were frozen separately in

liquid nitrogen and stored at -80�C. Homogenization

Fig. 1 Vectors and expression cassettes used in transformation

experiments, containing either the native (L1v) or the synthetic

(L1pt) transgene. For each vector, all regulatory sequences and

the accumulation of L1 protein in corresponding transplastomic

plants are indicated

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123

was performed by adding to the frozen samples (about

250,000 protoplasts each) 2 volumes of ice-cold 1.59

protoplast homogenization buffer (150 mM Tris–HCl,

150 mM NaCl, 1.5 mM EDTA, and 1.5% Triton

X-100, pH 7.5) supplemented, immediately before

use, with 1.5 mM phenylmethylsulfonyl fluoride and

the Complete (GE Healthcare) protease inhibitor

cocktail. The samples were used for immunoprecipi-

tation using the Tetra His antibody (Qiagen) and the

Mouse monoclonal anti-L1 antibody (Camvir 1, Mil-

lipore), according to manufacturer’s instructions,

followed by affinity selection with Protein-G agarose

(Pierce). Radioactive samples were analyzed by

SDS-PAGE on 15% acrylamide gels. Rainbow14C-methylated proteins (GE Healthcare) were used

as molecular weight markers. Gels were treated with

2,5-diphenyloxazole dissolved in DMSO, and radio-

active polypeptides were revealed by autoradiography.

Capture ELISA

Crude soluble extracts were obtained homogenizing

leaves in high salt buffer (19 PBS, 0.5 M NaCl,

10 mM EDTA, 1 mM PMSF buffer). The extracts

were centrifuged at 13,000 r.p.m. for 5 min and

supernatant was collected. Microtiter plates were

coated overnight with a 1:500 dilution of the protein-

A-purified mouse monoclonal antibody (50 ll/well)

Ritti1 (Muller et al. 1997), which recognizes a HPV-

16 L1 conformational epitope, then washed and

blocked for 1 h at 37�C in 5% milk PBS. Plant extract

was added at different dilutions, starting from 40 lg of

total proteins for each genotype, for 1 h at 37�C,

followed by three washing steps. The polyclonal rabbit

antiserum 4543 (1:3,000 dilution in 5% milk PBS)

raised against HPV-16 VLPs (kindly provided by Dr.

M. Muller, Heidelberg, Germany) was added, and

plates were incubated at 37�C for 1 h. Plates were

washed again and incubated with a goat-anti rabbit

peroxidase conjugate (1:5,000, GE Healthcare) for 1 h

at 37�C. After three washing steps, TMB substrate

(Biorad) was added and allowed to develop for

10 min. Baculovirus-derived VLPs of known concen-

trations (125–0.977 ng) were used as standards.

Electron microscopy

Plant proteins from control PRV111A and PL61

transplastomic plants were extracted using 19 PBS

buffer. Samples were viewed after immunotrapping

the particles with the monoclonal anti-L1 antibody

specific for HPV-16 VLPs (Muller et al. 1997) on

carbon coated grids, using the Immuno Sorbent

Electron Microscopy (ISEM) method (Milne and

Luisoni 1977). The grids were negatively stained with

2% uranyl acetate (Nermut 1982) and viewed using a

Philips Morgagni electron microscope at 80 kV.

Results

Vector construction and production of L1

transplastomic plants

To transform tobacco plastids, 10 vectors containing

either the native viral (L1v) or a synthetic (L1pt) L1

gene were constructed (Fig. 1). In the synthetic gene,

the codon usage was optimized for expression in

plastids, overall changing about one fourth of nucle-

otides. In addition, the last 22 amino acids of the

coding sequence, including a Nuclear Localization

Signal (NLS), were deleted and two aspartic acid

residues were added to the 30 end of the coding region

to prevent potential triggering of degradation by the

SsrA system, as observed for some proteins in E. coli

(Keiler et al. 1996; Hayes et al. 2002).

The transgenes were expressed in the strong rrn

operon promoter and the rbcL terminator cassettes.

The rrn promoter in most constructs was fused with

the plastid rbcL gene 50-untranslated region (50-UTR), as in plasmids pPL22, pNS31, pPL62, pPL63,

and pPL70, or 50-translation control region (TCR)

that includes the 50-UTR and 42 N-terminal nucleo-

tides of the rbcL open reading frame (plasmids pPL61

and pPL66). We also expressed L1 from chimeric

promoters with the atpB 50-TCR, including the

50-UTR and 42 N-terminal nucleotides of the atpB

open reading frame (plasmid pPL20), or the psbA and

T7 phage gene 10 50-UTRs (plasmids pPL24 and

pPL69, respectively). For purification of the recom-

binant L1 proteins either Gluthatione S-Transferase

(GST) or 6xHis tags and factor Xa protease recog-

nition sequence were added, as indicated in Fig. 1.

The transgenes were cloned in plastid pPRV vectors

(Zoubenko et al. 1994) targeting exogenous DNA to

the trnV-rps12/7 region of the tobacco plastid genome.

Transformation of tobacco plastids was carried out

with vectors listed in Fig. 1. Southern blot analysis

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with the targeting sequence (P1) probe confirmed

integration of foreign DNA in each of the clones

yielding the predicted hybridizing fragments: 4.6 and

2.2 kb in transgenic lines transformed with plasmid

pPL20, 6.4 kb in lines transformed with plasmid PL61

and PL66 and 3.3 kb in wild-type N. tabacum plants

(Fig. 2). Transplastomic seeds germinated on specti-

nomycin-containing medium were uniformly green

indicating that the transplastomic lines were homo-

plastomic (data not shown). Homoplasmic plants were

obtained for each of the lines listed in Fig. 1. Since L1

protein accumulated at detectable levels in only the

PL20, PL61 and PL66 plants, DNA gel blot analyses

data are shown here only for these lines.

Accumulation of L1 mRNA

To verify the accumulation of the recombinant

transcripts, we extracted total cellular RNA from

tobacco leaves and tested mRNA levels on RNA gel

blots using either the L1v or L1pt coding sequence

probe (Fig. 3). L1v transcripts were detected in PL20,

PL22 and PL24 transplastomic plants. Because of the

inefficient transcription termination by TrbcL, as

usually is the case with plastid terminators (Maliga

2002), plants showed both read-through dicistronic

(GST-L1/aadA) and monocistronic (GST-L1) tran-

scripts of about 4.1 (3.9 in PL24 lines) and 2.8 kb,

respectively (Fig. 3a). Probing the same blots with

the aadA gene confirmed that the longer transcript

contained sequences derived from both the GST-L1

and the aadA genes (data not shown). As regards L1pt

transplastomic plants, transcripts of 3.3 and 2.0 kb

were detected in PL61 and PL66 lines, and of 2.5 and

1.3 kb in PL63 plants (Fig. 3b). Abundant transcripts

of the expected size were obtained in all tested lines,

except for pPL62-derived plants, which displayed

low recombinant mRNA levels. Because the L1-gene

Fig. 2 Southern blot

analysis of representative

homoplasmic

transplastomic plants

transformed with the pPL20

(L1v), pPL61 and pPL66

(L1pt) vectors. Total cellular

DNA was digested with the

BamHI restriction enzyme

and probed with a P32

labelled ApaI–BamHI

targeting region fragment

(P1). Nt = N. tabacumwild-type; A = ApaI;

B = BamHI

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123

in PL62 plants is expressed in the same promoter-

terminator cassette as the others with L1pt gene, low

mRNA levels are likely due to accelerated mRNA

turnover.

Accumulation of recombinant L1 protein

Recombinant L1 protein accumulation was tested in

total soluble protein extracts from leaves expressing

L1 from several constructs, including a C-terminal

largely-deleted L1 (DC205 in pPL63) to exclude

potential negative effects associated with the protein

C-terminus and the L1 assembling process. Similar

C-terminal deletions were tested in the baculovirus

expression system, showing no VLPs or capsomeres,

but only protein aggregates (Touze et al. 2000).

Using a monoclonal anti-L1 antibody in Western blot

analyses, the recombinant protein of the expected size

was detected only in PL20 (82 kDa), PL61 and PL66

(55 kDa) lines (Fig. 4). No signal was obtained in

PRV111A-4A negative control (transgenic plants

transformed with aadA only) and in other L1

transplatomic lines (data not shown). Based on

dilution series of baculovirus-derived purified VLPs

as standards in Western blots, PL61 and PL66 lines

accumulated up to 1.5 and 0.8% of total proteins,

respectively, while PL20 leaves contained only up to

0.1% of total soluble leaf protein. In the Nt-PL20

extract, in addition to the 82 kDa band GST-L1

fusion protein, a 55 kDa L1 and a smaller band were

also present, suggesting posttranslational processing

of the 82 kDa protein encoded in the pPL20 gene.

Proteolytic degradation products were also observed

when HPV L1 proteins were produced in vaccinia

virus vectors, in fission yeast and from tobacco

nuclear genes (Hagensee et al. 1993; Sasagawa et al.

1995; Kohl et al. 2007).

Because of the superior immunogenicity of L1

multimeric structures (Biemelt et al. 2003; Varsani

et al. 2003; Dell et al. 2006; Thones and Muller 2007),

we tested VLP/capsomere accumulation in leaf protein

Fig. 3 Northern blot analysis of representative PL20, PL22,

PL24, PL61, pPL62, PL63, and PL66 transplastomic plants,

using specific probes for either L1v (A) or L1pt (B) gene. RNA

of 5 lg were loaded per lane. The schemes show the origin of

monocistronic (*) and dicistronic read-through (**) transcripts.

In PL24 lines, a shorter dicistronic transcript was expected due

to the absence of the terminator and promoter sequences

between GST-L1v and aadA genes. In PL63 lines, shorter

transcripts were due to the 615 bp deletion at the 30 end of the

L1 gene. As load controls, EtBr-stained total leaf RNAs are

reported in lower panels. Nt = N. tabacum wild-type

Fig. 4 Western blot analysis of PL61-3BA, PL66-4AA and

PL20-2A5 transplastomic plants, using an anti-L1 monoclonal

antibody (Camvir 1, Millipore). The PRV111A-4A line,

transformed with the empty vector pPRV111A, was used as

negative control. The amount of total proteins loaded per lane

is indicated. A dilution series of purified baculovirus-derived

L1 protein (L1 VLPs) was loaded as reference

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123

extracts. A capture ELISA assay on crude extracts, using

the Ritti1 monoclonal anti-L1 antibody that recognizes

conformational epitopes (Muller et al. 1997), showed

that the recombinant L1 protein was able to self-

assemble into higher-order structures in transplastomic

lines, when compared to the PRV111A-4A negative

control (Fig. 5). Significant differences, partially

reflecting those already observed in protein blots, were

obtained between the three transplastomic genotypes.

The assembling of L1 in complex structures was

confirmed by electron microscopy analysis. After im-

munotrapping of plant protein extracts using the same

anti-L1 monoclonal antibody, distinct spherical parti-

cles of about 55 nm and smaller, suggesting

the presence of VLPs and other L1-derived superstruc-

tures (including capsomeres), were detected in PL61

plants, but not in the pPRV111A-derived control plant

(Fig. 6).

Pulse-chase experiments to determine L1 protein

half-life

L1 stability and half-life was also studied in pulse-

chase experiments. A 35S-labelled protein of the

expected molecular weight (55 kDa), was detected in

PL66-4AA samples after 1 h pulse and immunose-

lection with anti-histidine antibody. The signal did

not markedly decrease during 8 h chase, confirming

that the protein was relatively stable in the plastid

environment. However, the signal was markedly

reduced by 24 h, suggesting a half-life between the

8 and 24 h chase points. When the same immuno-

precipitation procedure was carried out using anti-L1

antibody, the initial radioactive signal corresponding

to L1 was stronger compared to that revealed by the

anti-His antibody, but during the chase time the

signal decreased more rapidly (Fig. 7). These results

suggest that modifications in the conformation of the

protein occurring during the chase mask the epitope

recognized by the anti-L1 antibody. Two additional

cross-reactive polypeptides were immuno-precipi-

tated with anti-L1 antibodies both in PL66-4AA

and control PRV111A-4A plants.

Discussion

We report here the feasibility of producing HPV-16

L1 vaccine by expressing the L1 protein from the

native viral (L1v gene) or a synthetic sequence (L1pt

gene) optimized for expression under control of

plastid expression signals in tobacco chloroplasts. We

could detect L1 antigen accumulation only when the

Fig. 5 Accumulation of L1 VLPs and capsomeres in leaf

tissue extracts of PL61-3BA, PL66-4AA and PL20-2A5

transplastomic plants, as determined by capture ELISA with

an anti-L1 antibody which recognizes a conformational epitope

(Ritti1) (Muller et al. 1997). PRV111A-4A = negative con-

trol. Bars indicate SE of means

Fig. 6 Electron microscopy analysis carried out on leaf tissue

extracts from PRV111A-4A (negative control) and PL61-3BA

transplastomic plants, after immunotrapping the particles on

carbon coated grids with the Ritti1 monoclonal anti-L1

antibody (Muller et al. 1997). Some representative VLPs of

about 55 nm are indicated by the black arrows

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N-terminus of L1 protein was translationally fused

with the first 14 amino acids of the N-terminal

domain of the ATPase beta subunit or the Rubisco

large subunit. Fusion with the segment derived from

the plastid genes may facilitate protein accumulation

by efficient loading of the chimeric mRNAs onto the

plastid ribosomes or protecting the recombinant L1

protein from degradation. The finest example in this

respect was provided by the comparison between

PL61 and PL62 lines, that differed with respect to the

presence of the rbcL downstream sequence, with

significant (1.5%) L1 protein accumulation from the

promoter with the rbcL 50-TCR and no detectable

protein from the rbcL 50-UTR. Previously, the same

TCRs derived from the rbcL and atpB genes

increased NPTII translation accumulation from 4.7

to 10.8% and from 2.5 to 7% TSP, respectively

(Maliga 2002). The importance of the right fusion

segment is also indicated by the lack of protein

accumulation from fusions with GST (PL22 and

PL24 lines) and His-Xa sequence (NS31, PL62,

PL63, PL69, and PL70 lines). Based on ELISA and

electron microscopy analyses, the N-terminal fusions

of plastid-expressed proteins did not seem to prevent

L1 folding and higher-order structure formation,

similarly to what reported for GST-L1 fusion poly-

peptides expressed in bacteria, which folded and

formed pentamers properly (Chen et al. 2001).

Variability for L1 protein accumulation in trans-

plastomic plants did not seem to depend on differences

at the mRNA level, since, in Northern blot analyses

with specific L1v and L1pt probes, transcripts of the

expected size were detected in similar amounts with all

vectors, with the exception of the PL62 line, which

contained significantly lower L1 mRNA levels. The

only difference between pPL62 and comparable con-

structs with much higher mRNA levels (e.g. pPL61) is

the presence of the His-Xa sequence downstream of the

AUG. Similar destabilization of the mRNA was shown

in Nt-pHK39 plants due to manipulation of sequences

downstream of the AUG (Kuroda and Maliga 2001b).

The N-terminal fusion of L1 protein might affect

both the translatability of the mRNA and protein

stability. Results of Western blots suggest a higher

stability of the fusion protein derived from pPL61/

pPL66, with the first 14 aa of the RbcL subunit at the

N-terminal end, than that from pPL20 (AtpB-GST-

L1). In the latter case, in fact, the synthesized protein

is probably first cleaved between GST and L1, and

then further processed after having lost the N-terminal

protection. Pulse-chase experiments showed that the

N-terminus-protected L1 protein (RbcL-L1) synthe-

sized in plastids has an intermediate stability, showing

a half-life of more than 8 h. For comparison, other

plastidial proteins, such as the RuBPCase enzyme,

shows a half-life up to 6–7 days (Simpson 1981),

whereas the photosystem II D1 protein has a half-life

of less than 1 h (Whitney and Andrews 2001), the

latter showing rapid turnover and degradation prod-

ucts after the first minutes of synthesis (Kim et al.

1994). Several reports showed that the production of

recombinant pharmaceutical proteins in transgenic

plastids is highly dependent on light and develop-

mental status of plants (Birch-Machin et al. 2004;

Molina et al. 2004; Wirth et al. 2006), generally

showing higher proteolytic degradation and protein

instability in older leaves and under light. In the

present study, in order to test all different constructs

and to identify the optimized expression system,

experiments have been performed on in-vitro grown

plants. Our preliminary data indicate that the accu-

mulation of the L1 protein in greenhouse-grown

transplastomic plants decreases with leaf age (Z.

Adam, Rehovot, Israel, personal communication). A

detailed investigation on factors affecting L1 protein

stability in transgenic plastids is under way.

Low protein accumulation was obtained in early

experiments when the HPV-16 L1 protein was pro-

duced in nuclear transgenic plants (Biemelt et al. 2003;

Varsani et al. 2003; Liu et al. 2005). Recently, high

Fig. 7 Pulse-chase analysis of the L1 protein. The PRV111A-

4A transplastomic line was used as negative control. Protop-

lasts of PRV111A-4A and transplastomic PL66-4AA lines

were pulse-labelled for 1 h with 35S-Met and 35S-Cys and

chased for 0, 4, 8, 24 h as indicated. Immunoselection was

performed on protoplast homogenates using either anti-

histidine (Tetra Anti-His, Quiagen) or anti-L1 antibodies

(Camvir 1, Millipore). Analysis was by SDS-PAGE and

fluorography. The immunoprecipitated L1 protein (55 kDa) is

shown. Asterisks indicate aspecific immunoselected proteins

Transgenic Res

123

yield of L1 was reported from nuclear genes (Maclean

et al. 2007). Key factors for increasing protein accu-

mulation were the use of a proper 50-UTR, a human

codon usage-optimized sequence and a N-terminal

sequences targeting the L1 protein to the endoplasmic

reticulum or to the chloroplast. Of the two, the

chloroplast compartment accumulated higher levels

(up to 11%) of L1 protein. Higher levels of protein

accumulation was also observed when the labile COPV

(canine oral papillomavirus) L1 protein was targeted to

chloroplasts (Azhakanandam et al. 2007). Increased

accumulation in the chloroplast may be the result of a

reduced protease activity, or the presence of protective

chaperones in this cellular compartment.

It is noteworthy that all the constructs with the

plastid-optimized L1 gene, including those without

the N-terminal fusion, accumulated L1 protein in E.

coli (data not shown). These data are in agreement

with the results of previous studies indicating that a

presequence (protecting the L1 N-terminus) was not

necessary to accumulate the L1 protein in bacterial

cells (Zhang et al. 1998). These results suggest that

while the bacterial expression system is a potentially

useful tool to test plastid expression vectors, differ-

ences in mRNA turnover, translation and protein

degradation machineries of bacteria and higher plant

plastids make bacterial expression an unreliable

predictor of L1 protein expression in transplastomic

plants, as also reported for other recombinant proteins

(Magee et al. 2004).

Differences among the transplastomic lines were

observed in the Western blot analysis as well as in the

ELISA using antibodies detecting a linear or a

conformational epitope, respectively. Such differ-

ences, however, varied in the two assays. The relative

lower ability to form multimeric structures shown by

PL66 plants, considering the total protein level

detected with the linear mAb in western blot in

comparison with other genotypes, might depend on

the C-terminal His tag, which could interfere with the

VLP assembling of the L1 protein, given the relevant

role of ‘‘invading’’ C-terminal arms in anchoring

neighboring pentamers (Modis et al. 2002). On the

other hand, the presence of the NLS in PL20 plants,

that could somehow promote the assembling process

(Touze et al. 2000; Varsani et al. 2003; Kohl et al.

2007), might be responsible for the relative higher

production of assembled structures. The L1 concen-

tration in plastids could be an additional factor

affecting VLP/capsomer production in the tested

genotypes, since it is known to drive the transition

from free protein to VLP in plant cells and other

expression systems (Biemelt et al. 2003; Varsani

et al. 2003; Kohl et al. 2006; Varsani et al. 2006).

Pulse-chase experiments carried out using the anti-

His or anti-L1 (which recognizes a linear epitope)

antibodies showed an apparent lower protein stability

with the latter. As a consequence of L1 folding and

assembling into higher-order structures, in fact, the

epitope recognized by the anti-L1 antibody could be

buried faster than the His tag, because of its internal

position. This would be consistent with the model

proposed by Modis et al. (2002), postulating that the C-

terminal arm of the molecule, important for interca-

psomere interaction, is exposed to the surface (Carter

et al. 2003). However, plant extracts might contain

monomeric/pentameric L1 as well as L1 assembled

into higher-order structures, and different antibodies

could enrich either one of these fractions. Li et al.

(1998) showed that L1 susceptibility to degradation is

highly dependent on its incorporation into VLPs, and

L1 inside VLPs is at least partially protected from

proteolytic digestion by trypsin. The anti His-tag Ab

might have a bias to VLPs which has probably higher

half-life compared to unassembled molecules, whereas

the Ab recognizing the linear epitope precipitates

unassembled L1 and gives a higher initial yield (maybe

because more L1 is present in linear form than

assembled into VLPs), but an apparently decreased

half-life due to higher susceptibility of the protein.

Independently on the possible explanation for the

differences in apparent stability measured using the

two antibodies, we can certainly conclude that the half-

life of the protein is at least 8 h.

More experiments with other plant lines and con-

formational antibodies are necessary to conclude to

what extent the different constructs promote or prevent

VLP-formation and affect protein stability in trans-

genic plastids. In this study, some critical factors for

expressing the HPV-16 L1 protein in transplastomic

plants were highlighted. Nevertheless, the results

obtained demonstrate the feasibility of the production

of L1 multimeric structures (VLPs and capsomeres)

via plastid transformation. Both structures induced

neutralizing antibodies against HPV and related virus,

showing also protection against CRPV in rabbit

(Biemelt et al. 2003; Varsani et al. 2003; Dell et al.

2006; Kohl et al. 2006; Thones and Muller 2007).

Transgenic Res

123

Hence, considering the potential advantages of plastid

expression for biopharming (Bock 2007; Murphy

2007; Verma and Daniell 2007), our results are an

important step towards the development of a plant-

based HPV vaccine.

Acknowledgements This research was partially supported by

grants from the Italian Ministry of Health F.S.N. 2003 and

Italian Ministry of Research DD 1105/2002. PL was the

recipient of a Ph.D. studentship of the University of Naples

‘‘Federico II’’; part of the studentship was spent at Rutgers

University. Anita Morgese and Alfonso Piccolo are gratefully

acknowledged for technical help. We thank Dr. M. Muller

(Deutsches Krebsforschungszentrum, Forschungsschwerpunkt

Angewandte Tumorvirologie, 69120 Heidelberg, Germany) for

providing anti-L1 monoclonal Mabs, rabbit anti-L1 polyclonal

serum and baculovirus-derived VLPs. PL thanks in particular

Dr. Z. Svab for stimulating discussions and guidance during his

stay at Rutgers University.

Note added in proof: During the revision process of this

paper, another manuscript on L1 production in transgenic

tobacco plastids has been published (Fernandez-San Millan

et al. 2008).

References

Azhakanandam K, Weissinger SM, Nicholson JS, Qu R,

Weissinger AK (2007) Amplicon-plus targeting technology

(APTT) for rapid production of a highly unstable vaccine

protein in tobacco plants. Plant Mol Biol 63:393–404

Biemelt S, Sonnewald U, Galmbacher P, Willmitzer L, Muller M

(2003) Production of human papillomavirus type 16 virus-

like particles in transgenic plants. J Virol 77:9211–9220

Birch-Machin I, Newell CA, Hibberd JM, Gray JC (2004)

Accumulation of rotavirus VP6 protein in chloroplasts of

transplastomic tobacco is limited by protein stability.

Plant Biotechnol J 2:261–270

Bock R (2007) Plastid biotechnology: prospects for herbicide

and insect resistance, metabolic engineering and molec-

ular farming. Curr Opin Biotechnol 18:100–106

Buonaguro FM, Tornesello ML, Salatiello I, Okong P, Buon-

aguro L, Beth-Giraldo E, Biryahwaho B, Sempala SD,

Giraldo G (2000) The Uganda study on HPV variants and

genital cancers. J Clin Virol 19:31–41

Carter JJ, Wipf GC, Benki SF, Christensen ND, Galloway DA

(2003) Identification of a human papillomavirus type 16-

specific epitope on the C-terminal arm of the major capsid

protein L1. J Virol 77:11625–11632

Chen XS, Casini G, Harrison SC, Garcea RL (2001) Papillo-

mavirus capsid protein expression in Escherichia coli:purification and assembly of HPV11 and HPV16 L1. J

Mol Biol 307:173–182

Clifford GM, Smith JS, Plummer M, Munoz N, Franceschi S

(2003) Human papillomavirus types in invasive cervical

cancer worldwide: a meta-analysis. Br J Cancer 88:63–73

Daniell H (2006) Production of biopharmaceuticals and vac-

cines in plants via the chloroplast genome. Biotechnol J

1:1071–1079

Daniell H, Streatfield SJ, Wycoff K (2001) Medical molecular

farming: production of antibodies, biopharmaceuticals

and edible vaccines in plants. Trends Plant Sci 6:219–226

de Villiers EM, Fauquet C, Broker TR, Bernard HU, zur

Hausen H (2004) Classification of papillomaviruses.

Virology 324:17–27

Dell K, Koesters R, Linnebacher M, Klein C, Gissmann L

(2006) Intranasal immunization with human papilloma-

virus type 16 capsomeres in the presence of non-toxic

cholera toxin-based adjuvants elicits increased vaginal

immunoglobulin levels. Vaccine 24:2238–2247

Fernandez-San Millan A, Ortigosa SM, Hervas-Stubbs S, Corral-

Martınez P, Seguı-Simarro JM, Gaetan J, Coursaget P,

Veramendi J (2008) Human papillomavirus L1 protein

expressed in tobacco chloroplasts self-assembles into virus-

like particles that are highly immunogenic. Plant Biotech-

nol J (in press). doi:10:1111/j.1467-652.2008.00338.x

Fischer R, Stoger E, Schillberg S, Christou P, Twyman RM

(2004) Plant-based production of biopharmaceuticals.

Curr Opin Plant Biol 7:152–158

Frazer IH (2006) HPV vaccines. Int J Gynaecol Obstet

94(Suppl 1):S81–S88

Hagensee ME, Yaegashi N, Galloway DA (1993) Self-assem-

bly of human papillomavirus type 1 capsids by expression

of the L1 protein alone or by coexpression of the L1 and

L2 capsid proteins. J Virol 67:315–322

Hayes CS, Bose B, Sauer RT (2002) Proline residues at the

C terminus of nascent chains induce SsrA tagging

during translation termination. J Biol Chem 277:

33825–33832

Keiler KC, Waller PR, Sauer RT (1996) Role of a peptide

tagging system in degradation of proteins synthesized

from damaged messenger RNA. Science 271:990–993

Kim J, Klein PG, Mullet JE (1994) Synthesis and turnover of

photosystem II reaction center protein D1. Ribosome

pausing increases during chloroplast development. J Biol

Chem 269:17918–17923

Kohl T, Hitzeroth II, Stewart D, Varsani A, Govan VA,

Christensen ND, Williamson AL, Rybicki EP (2006)

Plant-produced cottontail rabbit papillomavirus L1 protein

protects against tumor challenge: a proof-of-concept

study. Clin Vaccine Immunol 13:845–853

Kohl TO, Hitzeroth II, Christensen ND, Rybicki EP (2007)

Expression of HPV-11 L1 protein in transgenic Arabid-opsis thaliana and Nicotiana tabacum. BMC Biotechnol

7:56

Kuroda H, Maliga P (2001a) Sequences downstream of the

translation initiation codon are important determinants of

translation efficiency in chloroplasts. Plant Physiol

125:430–436

Kuroda H, Maliga P (2001b) Complementarity of the 16S

rRNA penultimate stem with sequences downstream of

the AUG destabilizes the plastid mRNAs. Nucleic Acids

Res 29:970–975

Leggatt GR, Frazer IH (2007) HPV vaccines: the beginning of

the end for cervical cancer. Curr Opin Immunol 19:232–238

Li M, Beard P, Estes PA, Lyon MK, Garcea RL (1998) In-

tercapsomeric disulfide bonds in papillomavirus assembly

and disassembly. J Virol 72:2160–2167

Liu HL, Li WS, Lei T, Zheng J, Zhang Z, Yan XF, Wang ZZ,

Wang YL, Si LS (2005) Expression of human

Transgenic Res

123

papillomavirus type 16 L1 protein in transgenic tobacco

plants. Acta Biochim Biophys Sin (Shanghai) 37:153–158

Lutz KA, Bosacchi MH, Maliga P (2006) Plastid marker-gene

excision by transiently expressed CRE recombinase. Plant

J 45:447–456

Ma JK, Drake PM, Christou P (2003) The production of

recombinant pharmaceutical proteins in plants. Nat Rev

Genet 4:794–805

Ma JK, Barros E, Bock R, Christou P, Dale PJ, Dix PJ, Fischer

R, Irwin J, Mahoney R, Pezzotti M, Schillberg S, Sparrow

P, Stoger E, Twyman RM (2005) Molecular farming for

new drugs and vaccines. Current perspectives on the

production of pharmaceuticals in transgenic plants.

EMBO Rep 6:593–599

Maclean J, Koekemoer M, Olivier AJ, Stewart D, Hitzeroth II,

Rademacher T, Fischer R, Williamson AL, Rybicki EP

(2007) Optimization of human papillomavirus type 16

(HPV-16) L1 expression in plants: comparison of the suit-

ability of different HPV-16 L1 gene variants and different

cell-compartment localization. J Gen Virol 88:1460–1469

Magee A, Horvath E, Kavanagh TA (2004) Pre-screening

plastid transgene expression in Escherichia coli may be

unreliable as a predictor of expression levels in chloro-

plast-transformed plants. Plant Sci 166:1605–1611

Maliga P (2002) Engineering the plastid genome of higher

plants. Curr Opin Plant Biol 5:164–172

Maliga P, Kuroda H, Corneille S, Lutz K, Svab Z (2001)

Chloroplasts for the production of recombinant proteins.

In: Proceedings of the 12th international congress on

photosynthesis, Brisbane, Australia, August 18–23, 2001

McLean CS, Churcher MJ, Meinke J, Smith GL, Higgins G,

Stanley M, Minson AC (1990) Production and charac-

terisation of a monoclonal antibody to human

papillomavirus type 16 using recombinant vaccinia virus.

J Clin Pathol 43:488–492

Milne RG, Luisoni E (1977) Rapid immune electron micros-

copy of virus preparations. Methods Virol 6:265–281

Modis Y, Trus BL, Harrison SC (2002) Atomic model of the

papillomavirus capsid. EMBO J 21:4754–4762

Molina A, Hervas-Stubbs S, Daniell H, Mingo-Castel AM,

Veramendi J (2004) High-yield expression of a viral

peptide animal vaccine in transgenic tobacco chloroplasts.

Plant Biotechnol J 2:141–153

Munoz N, Bosch FX, de Sanjose S, Herrero R, Castellsague X,

Shah KV, Snijders PJ, Meijer CJ (2003) Epidemiologic

classification of human papillomavirus types associated

with cervical cancer. N Engl J Med 348:518–527

Murashige T, Skoog F (1962) A revised medium for rapid

growth and bioassays with tobacco tissue culture. Physiol

Plant 15:473–497

Murphy DJ (2007) Improving containment strategies in bi-

opharming. Plant Biotechnol J 5:555–569

Muller M, Zhou J, Reed TD, Rittmuller C, Burger A, Ga-

belsberger J, Braspenning J, Gissmann L (1997) Chimeric

papillomavirus-like particles. Virology 234:93–111

Nermut NV (1982) Advanced methods in electron microscopy

of viruses. In: Howard CR (ed) New developments in

practical virology. Alan Liss, New York, pp 1–58

Pedrazzini E, Giovinazzo G, Bollini R, Ceriotti A, Vitale A

(1994) Binding of BiP to an assembly-defective protein in

plant cells. Plant J 5:103–110

Sasagawa T, Pushko P, Steers G, Gschmeissner SE, Hajiba-

gheri MA, Finch J, Crawford L, Tommasino M (1995)

Synthesis and assembly of virus-like particles of human

papillomaviruses type 6 and type 16 in fission yeast

Schizosaccharomyces pombe. Virology 206:126–135

Schiller JT, Lowy DR (2006) Prospects for cervical cancer

prevention by human papillomavirus vaccination. Cancer

Res 66:10229–10232

Simpson E (1981) Measurement of protein degradation in

leaves of Zea mays using [3H] acetic anhydride and triti-

ated water. Plant Physiol 67:1214–1219

Stanley MA (2006) Human papillomavirus vaccines. Rev Med

Virol 16:139–149

Stiekema WJ, Heidekamp F, Dirkse WG, van Beckum J, de

Haan P, ten Bosh C, Louwerse JD (1988) Molecular

cloning and analysis of four potato tuber mRNAs. Plant

Mol Biol 11:255–269

Thones N, Muller M (2007) Oral immunization with different

assembly forms of the HPV 16 major capsid protein L1

induces neutralizing antibodies and cytotoxic T-lympho-

cytes. Virology 369:375–388

Touze A, Mahe D, El Mehdaoui S, Dupuy C, Combita-Rojas

AL, Bousarghin L, Pierre-Yves Sizaret PY, Coursaget P

(2000) The nine C-terminal amino acids of the major

capsid protein of the human papillomavirus type 16 are

essential for DNA binding and gene transfer capacity.

FEMS Microbiol Lett 189:121–127

Varsani A, Williamson AL, Rose RC, Jaffer M, Rybicki EP

(2003) Expression of human papillomavirus type 16 major

capsid protein in transgenic Nicotiana tabacum cv. Xan-

thi. Arch Virol 148:1771–1786

Varsani A, Williamson AL, Stewart D, Rybicki EP (2006)

Transient expression of Human papillomavirus type 16 L1

protein in Nicotiana benthamiana using an infectious

tobamovirus vector. Virus Res 120:91–96

Verma D, Daniell H (2007) Chloroplast vector systems for

biotechnology applications. Plant Physiol 145:

1129–1143

Whitney SM, Andrews TJ (2001) The gene for the ribulose-1,

5-bisphosphate carboxylase/oxygenase (Rubisco) small

subunit relocated to the plastid genome of tobacco directs

the synthesis of small subunits that assemble into Rubisco.

Plant Cell 13:193–205

Wirth S, Segretin ME, Mentaberry A, Bravo-Almonacid F

(2006) Accumulation of hEGF and hEGF-fusion pro-

teins in chloroplast-transformed tobacco plants is

higher in the dark than in the light. J Biotechnol

125:159–172

Zhang W, Carmichael J, Ferguson J, Inglis S, Ashrafian H,

Stanley M (1998) Expression of human papillomavirus

type 16 L1 protein in Escherichia coli: denaturation,

renaturation, and self-assembly of virus-like particles in

vitro. Virology 243:423–431

Zhou J, Doorbar J, Sun XY, Crawford LV, McLean CS, Frazer

IH (1991) Identification of the nuclear localization signal

of human papillomavirus type 16 L1 protein. Virology

185:625–632

Zoubenko OV, Allison LA, Svab Z, Maliga P (1994) Efficient

targeting of foreign genes into the tobacco plastid gen-

ome. Nucleic Acids Res 22:3819–3824

Transgenic Res

123