RESEARCH

Production of Functional Native Human Interleukin-2in Tobacco Chloroplasts

Xing-Hai Zhang • Patricia Keating •

Xia-Wei Wang • Yi-Hong Huang • James Martin •

James X. Hartmann • Aimin Liu

Published online: 22 October 2013

� Springer Science+Business Media New York 2013

Abstract Interleukin-2 (IL-2) is an important T lym-

phocyte-derived cytokine in the mammalian immune sys-

tem. Non-native, recombinant IL-2 derived from

Escherichia coli is used widely in both medical research

and treatment of diseases. Recombinant human IL-2 gene

has been expressed in plant nuclear genomes, therefore it

can be spread to the environment through pollen. Fur-

thermore, all the plant-produced IL-2 reported thus far had

been attached with artificial tags or fusion proteins, which

may trigger unintended immunological responses and

therefore compromise its full utility as a medicine. To

expand the potential of using plant chloroplasts to produce

functional native human therapeutic proteins, we inserted

an engineered human interleukin-2 (hIL-2)-coding gene,

without any tags, into the chloroplast genome of tobacco

(Nicotiana tabacum L.). Partially purified hIL-2 protein

from the leaves of the transplastomic plants induced

in vitro proliferation of IL-2-dependent murine T lym-

phocytes. Our study demonstrates that plant chloroplasts

can serve as a bio-factory for production of an active native

human interleukin in a self-contained and therefore envi-

ronmentally safe manner.

Keywords Chloroplast � Gene containment �Genetic engineering � Human interleukin-2 (hIL-2) �Tobacco (Nicotiana tabacum)

Introduction

Plants are a rich source of numerous bioactive compounds

important to human health, and possess the genetic and

biochemical capacity of synthesizing a vast array of ben-

eficial products, such as pharmaceuticals and nutraceuti-

cals. Transgenic plants have increasingly become an

attractive alternative system to animal or microbial-based

systems for producing human/animal proteins [1, 2].

Foreign genes are routinely introduced into the nuclear

genome of plants, but chloroplast transformation technol-

ogy allows the transgene to be inserted into a predeter-

mined site of the chloroplast (plastid) genome. Both

transcription and synthesis of the transgene protein occur

inside the chloroplast, offering several advantages over the

conventional nuclear transformation [3–7]. For example, it

allows precise gene targeting through homologous recom-

bination with a high level of gene expression due to a high

copy number of the chloroplast genome (up to 10,000

copies per leaf cell, as compared to 1–4 gene copies for

most nuclear genes), without apparent gene silencing.

Confinement of gene products inside the organelle miti-

gates potentially deleterious effects of foreign proteins on

the transformed cells. More significantly, transgenes are

largely contained and transmission by pollen is very rare

due to maternal inheritance of chloroplasts in most crop

plants, therefore alleviating the risk of transgene spread to

the environment. A large number of foreign proteins have

been produced through chloroplast transformation, some

achieving the astounding accumulation of 30 % or higher

X.-H. Zhang (&) � P. Keating � Y.-H. Huang � J. Martin �J. X. Hartmann

Department of Biological Sciences, Florida Atlantic University,

777 Glades Road, Boca Raton, FL 33431, USA

e-mail: xhzhang@fau.edu

X.-W. Wang

Provincial Hospital, Shandong University, Jinan,

Shandong 250021, China

Y.-H. Huang � A. Liu

Jiangsu Academy of Agricultural Sciences, Nanjing,

Jiangsu 210014, China

123

Mol Biotechnol (2014) 56:369–376

DOI 10.1007/s12033-013-9717-x

of total soluble leaf protein [e.g., 8–11]. Although the

actual foreign protein yields in chloroplasts vary greatly

among different reports, the potential of chloroplasts as a

robust factory for protein production is well documented.

Interleukin-2 (IL-2), a member of the cytokine family, is

part of the immune signaling system in humans and other

mammals and a major T cell growth factor exclusively

produced by activated T cells [12–14]. IL-2 plays a vital

role in the proliferation of helper and cytotoxic T cells, and

is essential for most adaptive immune responses [15].

Therefore, IL-2 has many important medical applications:

as an immunotherapy drug for cancers and chronic viral

infections and as an adjuvant (booster) for vaccines, as well

as a reagent for in vitro T cell studies [16–22]. Proleukin, a

recombinant IL-2, has been approved for treating malig-

nant melanoma, renal cell cancer, and chronic viral infec-

tions. The commercial recombinant IL-2 is a non-native

form obtained from a genetically engineered modification

of the human gene produced from bacterial systems, which

can be expensive and are prone to contamination by

pathogens that may infect humans. Thus, a plant system for

production of a native human IL-2 (hIL-2) may be a

desirable alternative. The first attempt [23] used a trans-

genic tobacco cell suspension culture to express the entire

hIL-2 cDNA, producing up to 0.38 lg hIL-2/g cell calli or

0.08 lg hIL-2/mL culture medium. This study demon-

strated the capability of plant cells to produce biologically

active hIL-2 as it supported the in vitro proliferation of the

IL-2-dependent murine CTLL-2 cell line. Another study

expressed the hIL-2-coding sequence (presumably also its

precursor form) in potato tuber and reported hIL-2 activity

equivalent up to 115 ng hIL-2/g potato tuber [24]. A more

recent effort [25] generated various lines of transgenic

tobacco plants that expressed mature hIL-2 or its fusions

with two different proteinase inhibitors. The 69 histidine-

or c-myc-tagged recombinant proteins were targeted to the

endoplasmic reticulum (ER), using a plant C-terminal ER-

retention signal peptide. A yield of 1.8–9.5 lg hIL-2/g

fresh leaf and 0.4–3.5 lg hIL-2-proteinase inhibitor fusion

protein/g fresh leaf was reported. Interestingly, both forms

of hIL-2 were shown to be active in CTLL-2 cell prolif-

eration, suggesting that attachment of poly-histidine,

c-myc, or proteinase inhibitor fusion proteins did not affect

IL-2 activity in vitro. Similarly, 69 histidine- or c-myc-

tagged hIL-2 protein that was targeted to the ER and

chloroplasts were expressed in transgenic tobacco plants

[26]. Whereas the yield was not measured, the purified hIL-

2 exhibited activity on murine splenic CD4? T cells.

Thus far all reported plant-based hIL-2 syntheses were

achieved via Agrobacterium-mediated nuclear transforma-

tion. One concern associated with the nuclear transforma-

tion is that the transgene can be spread to the environment

by pollen. Also, the reported tagged or protein-fused

recombinant hIL-2 may potentially compromise its down-

stream full utility as an immunological medicine. To our

knowledge, there is no report describing the expression of

any interleukin protein following chloroplast transforma-

tion. To explore the feasibility of plant chloroplasts as a

bioreactor for IL-2 synthesis, we report our efforts at

inserting the hIL-2 gene into the chloroplast genome,

allowing synthesis of the mature form of hIL-2 devoid of

any artificial tags. We investigated whether the organelle

gene expression apparatus was able to synthesize a func-

tional hIL-2 and whether the hIL-2 gene was contained

through maternal inheritance to prevent the chance of

dispersal via pollen that is an inherent concern with nuclear

transformation. Our studies demonstrate the utility of plant

chloroplasts as a self-contained bioreactor for the produc-

tion of native, active human hIL-2.

Materials and Methods

Chloroplast Transformation Vector for Human

Interleukin-2 (hIL-2) Synthesis

Oligonucleotides (primers) were purchased from Integrated

DNA Technologies (Coralville, IA). Primer J1A, 50-ACCATGGCACCTACTTCAAGTTCTACA-30, is located

in the beginning of the mature hIL-2 at Ala21 with addition

of a start codon (underlined) and a NcoI site. Primer J2, 50-AAGGTACCATACATTCAACAATAAATATAA-30, is

located 85 bp downstream of the stop codon of hIL-2 with

a KpnI site.

Primers J1A and J2 encompass the 402-bp-coding

region for the mature hIL-2 protein (Ala21–Thr153) that is

the native form of hIL-2 and the 85-bp 30-noncoding

sequence. PCR was carried out using the plasmid K1677

[American Type Culture Collection (ATCC), Manassas,

VA] containing the entire hIL-2 cDNA (GenBank acces-

sion no. BC066254) as template, and a high fidelity proof-

reading DNA polymerase Pfx (Invitrogen, Grand Island,

NY). The amplified fragment of 500 bp was ligated to

pCR�-Blunt II-TOPO (Invitrogen) and transferred into

Escherichia coli One Shot� TOP10 competent cells

(Invitrogen). The hIL-2 DNA plasmid was verified by

sequencing and fused with the tobacco chloroplast 16S

rRNA operon promoter Prrn [4] and the 30-untranslated

region of tobacco chloroplast ribosomal protein S16 as

terminator Trps16 [27]. The gene unit of Prrn::hIL-

2::Trps16 was then inserted into the StuI site of plasmid

pFaadA that contains the spectinomycin resistance gene

aadA (Fig. 1) [4]. Upon introduction into the chloroplasts,

hIL-2 and aadA genes were inserted into the intergenic

region between ndhF and trnL of the tobacco chloroplast

genome through homologous recombination (Fig. 1).

370 Mol Biotechnol (2014) 56:369–376

123

Chloroplast Transformation

Surface-sterilized seeds of tobacco (Nicotiana tabacum L.

cv. Petit Havana SR1) were grown aseptically on agar-

solidified medium [4] in a growth chamber at 24 �C under

*170 lmol quanta m-2 s-1 Gro-Lux fluorescent light

(16 h daily). Bombardment with DNA-coated gold parti-

cle (0.6 lm in diameter; Bio-Rad, Hercules, CA) of young

leaves and subsequent shoot regeneration under the

selection with spectinomycin (500 mg/L) were carried out

as previously described [4, 28–31]. The initial spectino-

mycin-resistant green shoots were screened by PCR to

detect the insertion of the hIL-2 gene at the expected site

of the genome. Leaves of the hIL-2 transplastomic shoots

were subjected to two more rounds of shoot regeneration

under spectinomycin selection until homoplasmy (i.e., all

copies of chloroplast genomes were inserted with the

transgenes) was confirmed by Southern blot analysis.

Resistant shoots were transferred to rooting medium

containing spectinomycin (500 mg/L). The rooted plants

were then transplanted to potting soil and grown to

maturity [4, 28, 29]. The seeds were collected and ger-

minated in medium containing spectinomycin (500 mg/L)

to verify 100 % resistance, typical of organelle genes

devoid of the Mendelian segregation associated with

nuclear genes. The soil grown plants were used for hIL-2

analysis.

In order to distinguish any possible effects of the

transformation vector, the pFaadA vector without hIL-2

was used to generate transplastomic plants for comparison.

Maternal Inheritance Analysis

Reciprocal crosses were performed as described in [29].

Seeds were collected and germinated on MS medium

containing 500 mg/L spectinomycin for 4 weeks before

scored for green or white seedlings.

DNA, RNA, and Protein Analysis of Transplastomic

Plants

Total DNA was isolated from leaves using DNeasy plant mini

kit (Qiagen, Valencia, CA). PCR was performed using prim-

ers L26a (50-ACTGGAAGTGGAATGAAAGGTATGA-30,286 bp upstream of the StuI site, Fig. 1) and J2 to verify the

insertion of hIL-2 between ndhF and aadA (Fig. 1). Southern

blot analysis was carried out with restriction enzymes-diges-

ted leaf DNA and probed with alkaline phosphatase (AP)-

labeled hIL-2 or 50-trnL DNA. Chemiluminescent detection

was done according to the manufacturer’s protocol (GE

Healthcare Bio-Sciences Corp, Piscataway, NJ).

Total RNA was isolated from young leaves at the same

developmental age of transplastomic and wild type plants

using Qiagen RNAeasy plant mini kit. Northern blot ana-

lysis was done using 20 lg RNA per sample with AP-

labeled hIL-2-coding sequence as probe, and using

chemiluminescent detection (GE Healthcare).

Total soluble proteins were extracted from same area of leaf

disks (1.8 cm2) from transplastomic and wild type plants. Leaf

disks were ground in liquid N2 to fine powder, homogenized in

protein extraction buffer [50 mM Tris–HCl, pH 7.5, 100 mM

NaCl, 5 mM EDTA, 1 mM DTT, 0.05 % Triton X-100,

complete protease inhibitor cocktail (Roche, Indianapolis, IN),

1 mg/mL polyvinylpolypyrrolidone], and centrifuged at

16,0009g, 4 �C for 10 min. Protein concentrations in the

supernatant were determined using a Bio-Rad protein assay kit,

with bovine serum albumin (BSA) as standard. Western ana-

lysis was done as previously described [29], using monoclonal

antibody against hIL-2 (Santa Cruz Biotechnology, Santa

Cruz, CA) and ECL detection reagents (Pierce, now Thermo

Scientific, Rockford, IL). E. coli derived recombinant human

IL-2 (R&D Systems, Minneapolis, MN) was used as standard.

Purification of hIL-2 from Transplastomic Plants

In order to remove compounds such as phenolics and

alkaloids that may be toxic to mammalian cell cultures, the

protein extract was partially purified by size chromatogra-

phy. Young leaves (*4–5 g fresh weight) were ground in

liquid N2 to fine powder and extracted with 5–10 mL of the

protein extraction buffer used above. After centrifugation at

16,0009g, 4 �C for 20 min, the clear supernatant fluid was

loaded onto a Bio-Spin 30 column (Bio-Rad) that was

Fig. 1 Site-specific integration of human IL-2 gene between ndhF

and trnL genes in tobacco chloroplast genome by homologous

recombination. The tobacco chloroplast 16S rRNA promoter (Prrn)

and ribosomal protein S16 terminator (Trps16) flank the mature hIL-

2-coding region (without its secretion signal sequence). Spectinomy-

cin resistance gene aadA (with Prrn as promoter and Chlamydomonas

rbcL 30-UTR as terminator) was used for selection for transformed

cell lines. Dashed lines delineate region of homologous recombina-

tion. Arrows indicate transcription direction. The location and

direction of primers L26a, J1A, and J2 are shown. The locations of

relevant restriction enzymes are also shown. The size is not to scale

Mol Biotechnol (2014) 56:369–376 371

123

prewashed with the extraction buffer. The protein extract

was eluted by gravity flow to the void volume at 4 �C. Then,

0.5–1 mL of cold PBS buffer (137 mM NaCl, 10 mM

Na2HPO4, 2 mM KH2PO4, 2.7 mM KCl, pH 7.5) was loa-

ded to the column and the elutes were collected. The elute

was freeze-dried, dissolved in 200 lL 0.1 % BSA in water,

sterilized with a 0.22 lm filter and stored at -80 �C until

used in the bioassays below.

Functional Bioassay of Chloroplast-Synthesized hIL-2

The activity of the chloroplast-derived hIL-2 was assayed

in vitro based on the effect of IL-2 on the proliferation of

murine CTLL-2 cytotoxic T cells [32]. The IL-2-dependent

CTLL-2 cell line (#TIB-214) was purchased from ATCC and

cultured in minimum essential medium (ATCC) supple-

mented with 10 % fetal bovine serum, 1 % penicillin/strep-

tomycin, 0.5 mM L-pyruvate, and 2 mM L-glutamine (all

from Sigma-Aldrich, St. Luis, MO) and 10 ng/mL E. coli-

produced recombinant hIL-2 (R&D Systems). Cultures were

placed in an incubator at 37 �C under 5 % CO2. When cell

cultures reached log phase, cells were plated in 96-well plates

(0.2 mL/well) at a density of 1.5 9 105 cells/mL in triplicate

wells, and incubated at 37 �C under 5 % CO2 for 4 days. Cells

were plated either in the presence of bacteria-derived hIL-2, or

protein extracts from transplastomic hIL-2 plants, a trans-

plastomic aadA vector-only plant and a wild type plant, or

complete media without added cytokine. The initial concen-

tration of bacterial-derived hIL-2 was 10 ng/mL, and the

initial concentration of plant hIL-2 extracts was a 1/1,000

dilution from the purified stocks. Five tenfold serial dilutions

of each protein were made and each of those concentrations

tested in triplicate. Following incubation at 37 �C for 4 days,

20 lL of Alamar blue (Life Technologies) was added to each

well and fluorescence determined using a fluorescence spec-

trophotometer at 560EX/590EM nm 24 h later.

A titration curve was established using nonlinear

regression analysis (Graph Prism 5.0 statistical software)

for the E. coli-derived recombinant hIL-2. The curves were

plotted as log [hIL-2] (ng/mL) on the x axis and OD560 nm

in the y axis. The log of the concentration for each plant

protein was interpolated from these titration curves. Trip-

licate curves and extrapolations were performed.

Results and Discussion

Transgene Construct Expressing Mature Human IL-2

in Tobacco Chloroplast

Precursor IL-2 produced by human-activated T cells is 153

amino acids long. The N-terminal 20-amino acid signal

peptide is cleaved between Ser20 and Ala21 during its

secretion process [33]. Therefore, the native and func-

tioning IL-2 produced by T lymphocytes is 133 amino

acids long. Unlike previous reports [23, 24], which

expressed the entire 153 amino acid hIL-2 precursor in

plants, we decided to use the native IL-2 coding sequence,

assuming no conceivable utility of the signal peptide in

plant cells. Furthermore, although attachment of tags or

fusion proteins to the IL-2 protein offers certain benefits,

we were concerned that any attachment might increase the

risk of unintended in vivo immune responses or toxicities

elicited by the tag acting as a foreign epitope or hapten in

humans when using IL-2 as a medicine. For example,

recombinant human TRAIL ligand with extraneous amino

acid residues showed hepatic toxicity while the native form

showed no toxicity in non-human primates [34, 35].

Therefore, in contrast to the previous reports [25, 26] of

plants expressing hIL-2 with histidine tag, c-myc tag and/or

proteinase inhibitor fusion proteins, we expressed the

mature hIL-2 gene without any tag. To achieve an active

gene expression in chloroplasts, a start codon (ATG) was

added to the mature hIL-2-coding sequence that was fused

to the chloroplast-specific promoter Prrn and terminator

Trps16 (Fig. 1). This Prrn::hIL-2::Trps16 gene unit, along

with selection gene aadA for spectinomycin resistance, was

flanked by anchoring sequences of ndhF and trnL at either

side. Thus, after the vector DNA was introduced into the

chloroplasts by particle bombardment, Prrn::hIL-2::Trps16,

along with aadA, was inserted into the intergenic region

between ndhF and trnL genes in the chloroplast genome

through homologous recombination (Fig. 1). Since the

complete sequence of the tobacco chloroplast genome is

known (GenBank accession no. Z00044), the gene target-

ing site can be readily determined by the calculated sizes of

specific restriction enzyme-digestion fragments.

Site-Specific Integration of hIL-2 Gene in Chloroplasts

Several independent transplastomic plants were generated.

These plants grew and reproduced normally. In order to

verify hIL-2 gene integration, Southern blot analysis was

carried out. When blots of HindIII-digested genomic DNA

isolated from different plants were probed with labeled

hIL-2 DNA, a 6.3 kb hybridizing band appeared in plants #

9, 10, and 12, but no band in both the wild type and plants

#7 and 11 that were transformed with an IL-2 free vector

(Fig. 2a), suggesting that plants # 9, 10, and 12 contained

the hIL-2-coding gene. The tobacco chloroplast genome

sequence predicts 3,669 bp between the nearest HindIII

site to ndhF gene and StuI (insertion site). Insertion of

*2.6 kb Prrn::hIL-2::Trps16-aadA between ndhF and trnL

(Fig. 1) should generate a 6.3 kb band in HindIII-digested

372 Mol Biotechnol (2014) 56:369–376

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DNA blots of plants # 9, 10, and 12 as observed in Fig. 2a,

indicating gene insertion at the predetermined site.

Likewise, digestion of the wild type chloroplast DNA with

MfeI or NdeI will produce fragments of 1,924 and 3,118 bp,

respectively, surrounding the regions between ndhF and trnL

as predicted from the genome sequence. As expected, blots

MfeI- or NdeI-digested wild type DNA hybridized with

labeled fragment of 50-trnL indeed showed a 1.9 kb band for

MfeI and a 3.1 kb band for NdeI (Fig. 2b). However, DNA

blots of plants # 9, 10, and 12 showed a *4.5 kb band for

MfeI and a *5.7 kb band for NdeI (Fig. 2b) due to the

insertion of *2.6 kb Prrn::hIL-2::Trps16 and aadA between

ndhF and trnL, again demonstrating site-specific integration

of the transgenes as specified in Fig. 1. A 3.4 kb band for

MfeI and 4.5 kb band for NdeI observed for plant #7 (Fig. 2b)

reflect the fact that this plant was generated from the aadA

vector without the *1.1 kb hIL-2 gene.

Furthermore, if any copies of the wild type chloroplast

genome remained in the transplastomic plants (hetero-

plastomy), hybridization of the DNA blots with a fragment

of the chloroplast gene trnL (Fig. 1) would reveal not only

the large band that contains hIL-2-aadA gene insert, but

also the smaller-sized wild type bands of 1.9 kb (MfeI) and

3.1 kb (NdeI). The absence of these bands in plants # 9, 10,

and 12 (Fig. 2b) indicates that these plants were homo-

plasmic, i.e., the hIL-2-aadA gene cassette was inserted

into all copies of the tobacco chloroplast genome.

Expression of hIL-2 Gene in Chloroplasts

When blots of total leaf RNA were hybridized to labeled

hIL-2, only transplastomic hIL-2 plants exhibited a

*0.8 kb band of hIL-2 mRNA, whereas there was no

signal from wild type (not shown) or vector-only plant

(Fig. 2c) because the hIL-2 gene is not expected to exist in

plants. It should be noted that the larger-sized faint band

appeared in the hIL-2 plants (Fig. 2c) probably resulted

from sporadic ‘‘read-through’’ of transcription that occurs

commonly in prokaryotic type of organelles such as chlo-

roplasts [4].

Western blot of crude leaf protein extracts using a

monoclonal hIL-2 antibody revealed an expected 15.5 kDa

protein in hIL-2 transplastomic plants, but not in the vec-

tor-only plant #7 (representative lines shown in Fig. 2d) or

the wild type plant (not shown). These results demonstrate

that these transplastomic plants produced the correct-sized

mature form of hIL-2 protein that was recognized by hIL-2

antibody as expected. To our knowledge, this is the first

report of chloroplast transgenic plants producing a human

interleukin.

It is well known that pollen cannot transmit organelle

DNA in tobacco. As expected (data not shown), our

transplastomic plants transmitted hIL-2 and aadA genes

maternally, not by pollen, which is advantageous to nuclear

transformation in terms of transgene containment.

Fig. 2 Integration and expression of hIL-2 gene in transplastomic

plants. Representative Southern blots with HindIII (a) and MfeI and

NdeI (b) show the site-specific insertion of hIL-2 gene in the

chloroplast genome of transplastomic plants # 9, 10, and 12. Northern

blot (c) shows the presence of hIL-2 mRNA in transplastomic plants #

9, 10, 12, and 21. Ethidium bromide-stained gel panel indicates RNA

loading. A representative western blot (d) using monoclonal anti-hIL-

2 antibody demonstrates the presence of hIL-2 in leaf protein extracts

from the same leaf area (1.77 cm2, 10–15 lg of total soluble protein).

E. coli-derived recombinant hIL-2 (10 ng) was used as a positive

reference. W untransformed wild type; hIL-2 transplastomic plants #

9, 10, 12, 13, and 21; plant #7: transformed with aadA-only vector

Mol Biotechnol (2014) 56:369–376 373

123

Function of Chloroplast-Synthesized hIL-2 in Murine

Cell Culture

The protein extracts from the control plants (either wild

type or aadA-only) had no activity on the proliferation of

murine IL-2-dependent CTLL-2 cells (Fig. 3). However,

protein isolated from the transplastomic hIL-2 plants

supported CTLL-2 cell proliferation, similarly to the

E. coli-derived hIL-2 (Fig. 3). Based on the activity curve

of known concentrations of the bacterial hIL-2, the amount

of chloroplast-synthesized hIL-2 was estimated to account

for *0.005 % of total soluble leaf protein, which is much

too low compared to some reports of other chloroplast-

synthesized recombinant proteins [7–11]. This yield is

somewhat comparable to that (0.002–0.05 % of total sol-

uble protein by our calculation) of the nuclear transgenic

hIL-2 tobacco plants reported [25]. However, low or even

undetectable levels of recombinant proteins have been

encountered in transplastomic plants for various reasons

[e.g., 4, 36–38]. Suboptimal translation of hIL-2 mRNA

and/or instability of hIL-2 protein may have caused the

low hIL-2 accumulation. For example, codon usage dif-

ference between a human gene and the protein synthesis

machinery in chloroplasts may affect translation effi-

ciency, even though no problem was reported for

expressing human somatotropin fused with ubiquitin in

chloroplasts [39, 40]. Intriguingly, fusion with two dif-

ferent proteinase inhibitors did not seem to enhance hIL-2

stability, instead it decreased its accumulation in the

transgenic tobacco leaves as compared to those without

inhibitors [25], indicating that IL-2 may be particularly

liable to degradation. The fact that recombinant hIL-2

yield is low in all the transgenic plants reported [23–25

and this study], whether nuclear or chloroplast transfor-

mation, suggests that a better design of transgene construct

focusing on translation efficiency such as codon optimi-

zation and stability [41] may be especially important for

small cytokine proteins like IL-2.

Interestingly, when the nuclear transgenic plants

expressed the human IL-10 fused to the transit peptide

sequence of Rubisco small subunit, IL-10 accumulated in

chloroplasts only if a His tag was added to the C-terminus

of IL-10, whereas His-tagged IL-10 did not accumulate in

mitochondria [42]. This study suggested that the C-termi-

nal His tag enhanced the stability of IL-10 in chloroplasts

but not in mitochondria. Therefore, the utility of artificial

tags in production of recombinant proteins needs more

careful evaluations, whether for their benefits of easy

detection and purification and increased stability, or for

concerns of unexpected, unwanted biological responses.

The decision of whether or not a tag/fusion protein is used

should be based on the property and purpose of individual

recombinant proteins of interest.

It is well known that post-translational modification can

be important to the function of proteins or enzymes. For

hIL-2, the disulfide bond between Cys58 and Cys105 is

necessary for biological activity [43]. Otherwise, it appears

that no specific modification is needed for hIL-2 activity, as

bacterial- or plant-derived recombinant hIL-2 seems bio-

chemically equivalent to the native human hIL-2. Particu-

larly interesting, the inclusion of the signal peptide at

N-terminus [23, 24], histidine and c-myc tags or proteinase

inhibitors at C-terminus [25, 26] does not appear to affect

IL-2 activity in vitro. Thus, as an in vitro assay reagent,

whether the IL-2 has a tag or not may not make much

difference. However, as aforementioned, it is conceivable

that extra attachments may change the functional config-

uration and therefore in vivo efficacy of IL-2. As an

immunotherapeutic cytokine, the presence of an attach-

ment on IL-2 protein may also trigger unintended immune

responses [34, 35], and therefore should be removed before

being used as a medicine. In any case, like bacterial sys-

tems, plants are capable of producing hIL-2 that is active

in vitro, regardless of whether the IL-2 gene is in the

nucleus or in the chloroplast.

Three other interleukins were reported to be produced in

transgenic plants, all via nuclear transformation and all

shown to be biologically active in vitro: human IL-4 in

tobacco cell suspension culture [23] and tobacco and potato

plants [44], human IL-10 and viral anti-inflammatory

cytokine IL-10 in tobacco plants [42, 45] and cell suspen-

sion cultures [46, 47] and Arabidopsis thaliana plants [48],

and mouse IL-12 (mIL-12) in tomato plants [49]. This

suggests that processing and post-translational modifica-

tions for these cytokines are compatible in the plant system.

In addition, mature forms of hIL-1b, hIL-7, hIL-10, mIL-4,

Fig. 3 Cell proliferation activity of recombinant hIL-2. A linear

regression curve (representative of three independent experiments)

was obtained by plotting log [hIL-2] (ng/mL) versus OD readings at

560 nm, for the E. coli-derived hIL-2 of known concentration (open

circle). The concentration of chloroplast-synthesized hIL-2 (closed

square) was extrapolated by plotting as OD560nm versus the logarithm

of five different 1/10 dilutions, starting from 1/1,000. The curve for

aadA vector-only plant (closed triangle) is also shown

374 Mol Biotechnol (2014) 56:369–376

123

and mIL-18, attached to the C-terminus His tag and ER

targeting, were synthesized in the endosperm cells of

transgenic rice plants [50]. The activity of these recombi-

nant interleukins in mammalian cells was not tested. It is

conceivable that chloroplast transformation technology can

also be used for production of these cytokines, taking

advantage of the high gene dosage and possible high levels

of expression of chloroplast genes as well as gene con-

tainment through maternal inheritance. Inclusion of a fusion

sequence or tag may improve protein synthesis and facili-

tate protein purification [37, 51, 52] but may also compli-

cate their use as a drug, unless the extraneous residues can

be removed efficiently. Therefore, optimization of gene

expression in bioengineered chloroplasts to enhance protein

synthesis and stability, along with cost-effective purifica-

tion without hindering protein integrity, should lead to

increased productivity of value-enhanced crops (e.g.,

tobacco) for pharmaceuticals, as an environmentally safer

alternative to animal or microbial cell cultures.

Acknowledgments We thank Elumalai Sivamani and Rongda Qu

for advice on protamine-using particle bombardment, Di Ding,

Michael Lucchese, Liz Barraco, Peter Stawinski, Devon Ghee, Efrain

(Alex) Negron, and Nickolas Skamangas for help with the particle

bombardment and tissue culture work. JM thanks Florida Atlantic

University (FAU) for an undergraduate research grant. This work was

supported in part by a fund from FAU to X.-H.Z.

References

1. Giddings, G., Allison, G., Brooks, D., & Carter, A. (2000).

Transgenic plants as factories for biopharmaceuticals. Nature

Biotechnology, 18, 1151–1155.

2. Ma, J. K.-C., Drake, P. M. W., & Christou, P. (2003). The pro-

duction of recombinant pharmaceutical proteins in plants. Nature

Review Genetics, 4, 794–805.

3. Bogorad, L. (2000). Engineering chloroplasts: An alternative site

for foreign genes, proteins, reactions and products. Trends in

Biotechnology, 18, 257–263.

4. Zhang, X.-H., Brotherton, J. E., Widholm, J. M., & Portis, A. R.,

Jr. (2001). Targeting a nuclear anthranilate synthase a-subunit

gene (ASA2) to the tobacco plastid genome results in enhanced

tryptophan biosynthesis. Return of a gene to its pre-endosymbi-

otic origin. Plant Physiology, 127, 131–141.

5. Daniell, H. (2006). Production of biopharmaceuticals and vac-

cines in plants via the chloroplast genome. Biotechnology Jour-

nal, 1, 1071–1079.

6. Maliga, P., & Bock, R. (2001). Plastid biotechnology: Food, fuel,

and medicine for the 21st century. Plant Physiology, 155,

1501–1510.

7. Hanson, M. R., Gray, B. N., & Ahner, B. A. (2013). Chloroplast

transformation for engineering of photosynthesis. Journal of

Experimental Botany, 64, 731–742.

8. De Cosa, B., Moar, W., Lee, S.-B., Miller, M., & Daniell, H.

(2001). Overexpression of the Bt cry2Aa2 operon in chloroplasts

leads to formation of insecticidal crystals. Nature Biotechnology,

19, 71–74.

9. Molina, A., Hervas-Stubbs, S., Daniell, H., Mingo-Castel, A. M.,

& Veramendi, J. (2004). High-yield expression of a viral peptide

animal vaccine in transgenic tobacco chloroplasts. Plant Bio-

technology Journal, 2, 141–153.

10. Oey, M., Lohse, M., Kreikemeyer, B., & Bock, R. (2009).

Exhaustion of the chloroplast protein synthesis capacity by

massive expression of a highly stable protein antibiotic. The Plant

Journal, 57, 436–445.

11. Lentz, E. M., Segretin, M. E., Morgenfeld, M. M., Wirth, S. A.,

Dus Santos, M. J., Mozgovoj, M. V., et al. (2010). High

expression level of a foot and mouth disease virus epitope in

tobacco transplastomic plants. Planta, 231, 387–395.

12. Smith, K. A. (1988). Interleukin-2: Inception, impact, and

implications. Science, 240, 1169–1176.

13. Xue, H.-H., Kovanen, P. E., Pise-Masison, C. A., Berg, M., Ra-

dovich, M. F., Brady, J. N., et al. (2002). IL-2 negatively regu-

lates IL-7 receptor a chain expression in activated T

lymphocytes. Proceedings of the National Academy of Sciences

of the United States of America, 99, 13759–13764.

14. Bachmann, M. F., & Oxenius, A. (2007). Interleukin 2: From

immunostimulation to immunoregulation and back again. EMBO

Reports, 8, 1142–1148.

15. Paul, W. E. (2008). Fundamental immunology. Philadelphia:

Lippincott-Williams & Wilkins.

16. Fyfe, G., Fisher, R. I., Rosenberg, S. A., Sznol, M., Parkinson, D.

R., & Louie, A. C. (1995). Results of treatment of 255 patients

with metastatic renal cell carcinoma who received high-dose

recombinant interleukin-2 therapy. Journal of Clinical Oncology,

13, 688–696.

17. Kammula, U. S., White, D. E., & Rosenberg, S. A. (1998). Trends

in the safety of high dose bolus interleukin-2 administration in

patients with metastatic cancer. Cancer, 83, 797–805.

18. Saparov, A., Wagner, F. H., Zheng, R., Oliver, J. R., Maeda, H.,

Hockett, R. D., et al. (1999). Interleukin-2 expression by a sub-

population of primary T cells is linked to enhanced memory/

effector function. Immunity, 11, 271–280.19. Lindsey, K. R., Rosenberg, S. A., & Sherry, R. M. (2000). Impact

of the number of treatment courses on the clinical response of

patients who receive high-dose bolus interleukin-2. Journal of

Clinical Oncology, 18, 1954–1959.

20. Fisher, R. I., Rosenberg, S. A., & Fyfe, G. (2000). Long-term

survival update for high-dose recombinant interleukin-2 in

patients with renal cell carcinoma. The Cancer Journal from

Scientific American, 6(Supplement 1), S55–S57.

21. Schwartzentruber, D. J. (2001). Guidelines for the safe adminis-

tration of high-dose interleukin-2. Journal of Immunotherapy, 24,

287–293.

22. Natarajan, V., Lempicki, R. A., Sereti, I., Badralmaa, Y., A-

delsberger, J. W., Metcalf, J. A., et al. (2002). Increased

peripheral expansion of naive CD4? T cells in vivo after IL-2

treatment of patients with HIV infection. Proceedings of the

National Academy of Sciences of the United States of America,

99, 10712–10717.

23. Magnuson, N. S., Linzmaier, P. M., Reeves, R., An, G., Hay-

Glass, K., & Lee, J. M. (1998). Secretion of biologically active

human interleukin-2 and interleukin-4 from genetically modified

tobacco cells in suspension culture. Protein Expression and

Purification, 13, 45–52.

24. Park, Y., & Cheong, H. (2002). Expression and production of

recombinant human interleukin-2 in potato plants. Protein

Expression and Purification, 25, 160–165.

25. Redkiewicz, P., Wiesyk, A., Gora-Sochacka, A., & Sirko, A.

(2012). Transgenic tobacco plants as production platform for

biologically active human interleukin 2 and its fusion with

proteinase inhibitors. Plant Biotechnology Journal, 10,

806–814.

26. Matakas, J. D., Balan, V., Carson, W. F., I. V., Gao, D., Bran-

dizzi, F., Kunkel, S., et al. (2013). Plant-produced recombinant

Mol Biotechnol (2014) 56:369–376 375

123

human interleukin-2 and its activity against splenic CD4?

T-cells. International Journal of Life Sciences Biotechnology and

Pharma Research, 2, 192–203.

27. Staub, J. M., & Maliga, P. (1994). Translation of psbA mRNA is

regulated by light via the 50-untranslated region in tobacco

plastids. The Plant Journal, 6, 547–553.

28. Zhang, X.-H., Ewy, R. G., Widholm, J. M., & Portis, A. R., Jr.

(2002). Complementation of the nuclear antisense rbcS-induced

photosynthesis deficiency by introducing an rbcS gene into the

tobacco plastid genome. Plant Cell & Physiology, 43, 1302–1313.

29. Zhang, X.-H., Webb, J., Huang, Y.-H., Lin, L., Tang, R.-S., &

Liu, A. (2011). Hybrid Rubisco of tomato large subunits and

tobacco small subunits is functional in tobacco plants. Plant

Science, 180, 480–488.

30. Barone, P., Zhang, X.-H., & Widholm, J. M. (2009). Tobacco

plastid transformation using the feedback-insensitive anthranilate

synthase [a]-subunit of tobacco (ASA2) as a new selectable

marker. Journal of Experimental Botany, 60, 3195–3202.

31. Sivamani, E., DeLong, R. K., & Qu, R. (2009). Protamine-

mediated DNA coating remarkably improves bombardment

transformation efficiency in plant cells. Plant Cell Reports, 28,

213–221.

32. Belani, R., & Weiner, G. J. (1996). Expression of both B7-1 and

CD28 contributes to the IL-2 responsiveness of CTLL-2 cells.

Immunology, 87, 271–274.

33. Taniguchi, T., Matsui, H., Fujita, T., Takaoka, C., Kashima, N.,

Yoshimoto, R., et al. (1983). Structure and expression of a cloned

cDNA for human interleukin-2. Nature, 302, 305–310.

34. Ashkenazi, A., Pai, R. C., Fong, S., Leung, S., Lawrence, D. A.,

Marsters, S. A., et al. (1999). Safety and antitumor activity of

recombinant soluble Apo2 ligand. Journal of Clinical Investiga-

tions, 104, 155–162.

35. Kelley, S. K., Harris, L. A., Xie, D., DeForge, L., Totpal, K.,

Bussiere, J., et al. (2001). Preclinical studies to predict the dis-

position of Apo2L/tumor necrosis factor-related apoptosis-

inducing ligand in humans: Characterization of in vivo efficacy,

pharmacokinetics, and safety. Journal of Pharmacology and

Experimental Therapeutics, 299, 31–38.

36. Whitney, S. M., & Andrews, T. J. (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. The

Plant Cell, 13, 193–205.

37. De Marchis, F., Pompa, A., & Bellucci, M. (2012). Plastid pro-

teostasis and heterologous protein accumulation in transplastomic

plants. Plant Physiology, 160, 571–581.

38. Caroca, R., Howell, K. A., Hasse, C., Ruf, S., & Bock, R. (2013).

Design of chimeric expression elements that confer high-level

gene activity in chromoplasts. The Plant Journal, 73, 368–379.

39. Staub, J. M., Garcia, B., Graves, J., Hajdukiewicz, P. T. J.,

Hunter, P., Nehra, N., et al. (2000). High-yield production of a

human therapeutic protein in tobacco chloroplasts. Nature Bio-

technology, 18, 333–338.

40. Maliga, P. (2002). Engineering the plastid genome of higher

plants. Current Opinion in Plant Biology, 5, 164–171.

41. Apel, W., Schulze, W. X., & Bock, R. (2010). Identification of

protein stability determinants in chloroplasts. The Plant Journal,

63, 636–650.

42. Menassa, R., Kennette, W., Nguyen, V., Rymerson, R., Jevnikar,

A., & Brandle, J. (2004). Subcellular targeting of human inter-

leukin-10 in plants. Journal of Biotechnology, 108, 179–183.

43. Wang, A., Lu, S. D., & Mark, D. F. (1984). Site-specific muta-

genesis of the human interleukin-2 gene: Structure–function

analysis of the cysteine residues. Science, 224, 1431–1433.

44. Ma, S., Huang, Y., Davis, A., Yin, Z., Mi, Q., Menassa, R., et al.

(2005). Production of biologically active human interleukin-4 in

transgenic tobacco and potato. Plant Biotechnology Journal, 3,

309–318.

45. Menassa, R., Nguyen, V., Jevnikar, A., & Brandle, J. (2001). A

self-contained system for the field production of plant recombi-

nant interleukin-10. Molecular Breeding, 8, 177–185.

46. Bortesi, L., Rademacher, T., Schiermeyer, A., Schuster, F., Pez-

zotti, M., & Schillberg, S. (2012). Development of an optimized

tetracycline-inducible expression system to increase the accu-

mulation of interleukin-10 in tobacco BY-2 suspension cells.

BMC Biotechnology, 12, 40. doi:10.1186/1472-6750-12-40.

47. Kaldis, A., Ahmad, A., Reid, A., McGarvey, B., Brandle, J., Ma,

S., et al. (2013). High-level production of human interleukin-10

fusions in tobacco cell suspension cultures. Plant Biotechnology

Journal, 11, 535–545.

48. Chen, L., Dempsey, B. R., Gyenis, L., Menassa, R., Brandle, J.

E., & Dhaubhadel, S. (2013). Identification of the factors that

control synthesis and accumulation of a therapeutic protein,

human immune-regulatory interleukin-10, in Arabidopsis thali-

ana. Plant Biotechnology Journal, 11, 546–554.

49. Gutierrez-Ortega, A., Sandoval-Montes, C., de Olivera-Flores, T.

J., Santos-Argumedo, L., & Gomez-Lim, M. A. (2005). Expres-

sion of functional interleukin-12 from mouse in transgenic tomato

plants. Transgenic Research, 14, 877–885.

50. Kudo, F., Ohta, M., Yang, L., Wakasa, Y., Takahashi, S., &

Takaiwa, F. (2013). ER stress response induced by the production

of human IL-7 in rice endosperm cells. Plant Molecular Biology,

81, 461–475.

51. Scotti, N., Alagna, F., Ferraiolo, E., Formisano, G., Sannino, L.,

Buonaguro, L., et al. (2009). High-level expression of the HIV-1

Pr55gag polyprotein in transgenic tobacco chloroplasts. Planta,

229, 1109–1122.

52. Elghabi, Z., Karcher, D., Zhou, F., Ruf, S., & Bock, R. (2011).

Optimization of the expression of the HIV fusion inhibitor cya-

novirin-N from the tobacco plastid genome. Plant Biotechnology

Journal, 9, 599–608.

376 Mol Biotechnol (2014) 56:369–376

123