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Int. J. Mol. Sci. 2016, 17(5), 624; doi:10.3390/ijms17050624

Article
Production of Bioactive Recombinant Bovine Chymosin in Tobacco Plants
Zheng-Yi Wei 1,2, Yu-Ying Zhang 2,3, Yun-Peng Wang 2, Ming-Xia Fan 1, Xiao-
Fang Zhong 2, Nuo Xu 4, Feng Lin 1,* and Shao-Chen Xing 2,*
1

Agronomy College, Shenyang Agricultural University, No. 120 Dongling Rd., Shenhe
Distirct, Shenyang 110866, China
2

Laboratory of Plant Bioreactor and Genetics Engineering, Jilin Provincial Key

Laboratory of Agricultural Biotechnology, Agro-Biotechnology Research Institute, Jilin
Academy of Agricultural Sciences, No. 1363 Shengtai St., Changchun 130033, China
3

State Key Labortory of Agrobiotechnology, China Agricultural University, No. 2 West

Yuanmingyuan Rd., Beijing 100094, China
4

Chashan Higher Education Zone, Wenzhou University, Wenzhou 325035, China

*
Correspondence: Tel.: +86-24-8848-7163 (F.L.); +86-431-8706-3121 (S.-C.X.); Fax:
+86-24-8848-7161 (F.L.); +86-431-8706-3080 (S.-C.X.)
Academic Editor: Marcello Iriti
Received: 24 February 2016 / Accepted: 14 April 2016 / Published: 28 April 2016
Abstract
: Chymosin (also known as rennin) plays an essential role in the coagulation of milk in
the cheese industry. Chymosin is traditionally extracted from the rumen of calves and is
of high cost. Here, we present an alternative method to producing bovine chymosin
from transgenic tobacco plants. The CYM gene, which encodes a preprochymosin from
bovine, was introduced into the tobacco nuclear genome under control of the viral 35S
cauliflower mosaic promoter. The integration and transcription of the foreign gene were
confirmed with Southern blotting and reverse transcription PCR (RT-PCR) analyses,
respectively. Immunoblotting analyses were performed to demonstrate expression of
chymosin, and the expression level was quantified by enzyme-linked immunosorbent
assay (ELISA). The results indicated recombinant bovine chymosin was successfully
expressed at an average level of 83.5 ng/g fresh weight, which is 0.52% of the total
soluble protein. The tobacco-derived chymosin exhibited similar native milk
coagulation bioactivity as the commercial product extracted from bovine rumen.
Keywords:
chymosin; rennin; tobacco; milk coagulation; transformation

1. Introduction
Chymosin, more commonly known as rennin, is a key industrial enzyme used to
produce cheese, a historically important food in Western countries and one that is in
increasing demand in Eastern countries such as China. Cheeses are distinguished by
their flavors and/or textures which depend both on their composition and the chymosin
used in cheese processing to coagulate the milk [1,2,3,4]. An enzyme with a high
clotting to proteolytic activity ratio (C/P value) is preferred because a high proteolytic
activity will lead to poorer flavor and/or texture. As bovine chymosin has a high C/P
value, it is extremely suitable for cheese production [5]. This is the main reason why
bovine chymosin is still widely used and often added to other sources of chymosin such
as microorganisms, plants, and other animals [6,7,8,9,10,11,12,13].
Given that chymosin production from calf rumen is limited, alternative methods are
needed to produce sufficient bovine chymosin to meet the increasing global demand.
The cDNA of the gene encoding bovine chymosin was cloned and analyzed in the early
1980s [14,15], and, since then, many efforts had been made to express recombinant
chymosin in various types of heterologous organisms. Successful expression has been
achieved in many species such as Escherichia coli [16], Bacillus species
[17], Aspergillus species [18,19,20,21], and yeast [22,23,24,25]. Animal-coded
chymosins now constitute more than 70% of the global chymosin market [26] with 80%
of fermentation chymosin produced as recombinant proteins in microorganisms [27],
with protein produced in Aspergillus and yeast the most widely used.
Numerous types of proteins including vaccines, antibodies, therapeutic proteins, and
industrial enzymes have been successfully expressed using plant expression systems
both in intact plants and in plant cell culture [28,29]. Compared with microorganisms,
expression of foreign proteins in plants has a number of advantages including superior
safety and scale-up capacity, better product quality, more accurate protein folding and
post-translational modification. Most importantly, expression in plants offers a low-cost
solution for production and storage [30,31,32]. Plant expression systems can be broadly
divided into two categories: stable and transient; with stable expression achieved by
either the transformation of the nucleus or the plastid, depending on the situation.
Rather surprisingly, given its importance as an industrial enzyme and its potential as a
therapeutic enzyme for some disease treatments [33], there have been limited attempts
to express chymosin in plants. Willmitzer et al. first reported the expression of
recombinant chymosin in tobacco leaves [34]. The second example was reported by van
Rooijen et al., who developed a seed expression system to achieve a higher expression
level [35].
In this paper, we report the expression of bovine chymosin in transgenic tobacco plants.
The expression level varied between the different plants, but the highest yield of
chymosin was determined to be 83.5 ng/g fresh weight (or 0.52% of total soluble
protein (TSP)). The plant-derived chymosin was still active and capable of clotting
milk. This study offers an alternatively approach to producing chymosin for cheese
production.

2. Results
2.1. Vector Construction and Tobacco Transformation
The bovine preprochymosin expression vector, p33cym11, was successfully
constructed. In Figure 1A, the preprochymosin gene is controlled by the cauliflower
mosaic virus 35S promoter (CaMV 35S) and the terminator of nopaline synthase gene
(Nos). In the vector, a phosphinothricin acetyltransferase gene, conferring resistance to
glufosinate, was used as the selectable marker in tobacco.

Figure 1. Generation of transgenic plants. (A) The T-DNA insert in the bovine
preprochymosin (CYM) expression vector p33cym11. LB and RB represent the left
and right border of T-DNA region; PPTR represents the glufosinate-resistance
encoding gene; CaMV 35S and 35S polyA represent the promoter and the terminator
of cauliflower mosaic virus 35S gene respectively; Lac Zα encodes the α-fragment of
the enzymy β-galactosidase; Nos polyA represents the terminator of the nopaline
synthase gene; (B) Resistant shoots regenerated on a selection medium; (C)
Resistant shoots rooting on rooting medium; (D) Plants grown in the greenhouse.
After wiping glufosinate solution on the circled portions of the leaves, no necrotic
lesions were detected for the transgenic plant (left) but obvious symptoms were
observed in the wild type (right). Scale bars = 1 cm.

Transgenic plants containing the preprochymosin gene were obtained

via Agrobacterium-mediated leaf disc transformation. The plants were generated on a
medium, plus 2 mg/L of glufosinate as a selectable reagent. The transgenic plants
displayed glufosinate resistance and showed a normal WT phenotype (Figure 1B–D).

2.2. Molecular Analyses

Genomic DNA was extracted from glufosinate-resistant plants to perform molecular
analyses. PCR analyses, carried out as a preliminary screen to confirm the existence of
the preprochymosin gene, CYM, revealed that most of the resistant plants were positive
(Figure 2A). The integration of the preprochymosin gene was further confirmed with
Southern blotting. The results demonstrated that all the plants tested were transgenic but
contained different copies of the foreign gene (Figure 2B). There was one copy in line
11J and two or more copies in other four lines. The RT-PCR results indicated that
the CYMgene was transcribed in all the tested plants (Figure 2C), but there is seemingly
no relation between the transcription level and copy number inserted. Line 11L, for
instance, had two copies of the target gene, but showed the lowest accumulation of
mRNA, while line 11J harboring a single copy was observed with a much higher
accumulation.

Figure 2. Molecular analysis of transgenic plants. (A) PCR analysis of the resistant
regenerated plants. M, DNA ladder (Transgene, Beijing, China, cat. No.BM301); +,
vector p33cym11 as positive control; --, H2O as blank control; CK, wild-type plant;
Lane 1-10, resistant plants; (B) Southern Blotting analyses using a CYM fragment as
probe to hybridize with DNAs digested by Bam HI and Hind III. M, DNA ladder
(lambda DNA digested by Sty I); +, vector p33cym11 as positive control; CK, wild-
type plant; 11A, 11C, 11I, 11J, and 11L are different transgenic lines; (C)
Transcription analysis of the CYM gene in transgenic plants. Cym indicates the
specific bands for the CYM gene, 5S rRNA shows the RNA amount used in the
analysis. 11I, 11A, 11J, 11L, and 11C are different transgenic lines; +, vector
p33cym11 as positive control; CK, wild-type plant.

2.3. Expression of Recombinant Bovine Chymosin and Detection of Activity

Immunoblotting confirmed the successful expression of bovine chymosin in TSP
extracted from the plants (Figure 3A). A quantitative analysis by ELISA indicated that
expression levels varied from 18.1 to 83.5 ng/g fresh weight, meaning 0.18% to 0.52%
of TSP (Figure 3B).
 Figure 3. Expression of recombinant bovine chymosin in transgenic tobacco plants.
(A) Immunoblotting analysis; (B) Yield of recombinant bovine chymosin in
transgenic plants. Chymosin, CK, and rProchymosin represent commercial chymosin
isolated from calf, crude TSP from wild-type plant and recombinant prochymosin
with 6× His tag from E. coli respectively; 11A, 11C, 11I, 11J, and 11L represent
different individual transgenic plant lines or crude TSP from them. Yield data are
collected from three independent duplicates for each line.

A bioactivity assay was performed to test whether the plant-derived chymosin was still
functional. In this assay, the time taken for fresh milk to be clotted by the plant TSP was
assessed. All plants expressing chymosin displayed natural bioactivity. As the
expression level varied in different transgenic plants, the crude chymosin exhibited
different levels of bioactivity, at a milk-clotting time ranging from 20.5 to 46.3 min
(Figure 4). Interestingly, the accumulation of mRNA possibly was correlated with the
yield of protein.
 Figure 4. Milk clotting tests for recombinant bovine chymosin. The status of milk
clotting (A) and the time consumed (B) are shown. Chymosin, PBS, and CK
represent the commercial chymosin from calf, PBS buffer, and crude TSP from wild-
type plant respectively; 11A, 11C, 11I, 11J, and 11L represent crude TSPs from the
transgenic plants accordingly. Data of clotting time are collected from three
independent repeats.

3. Discussion
Although bovine chymosin has been expressed in many microorganisms, production
still cannot meet market demands [36]. Expression of chymosin in plants might help
address this shortfall since the GM plant offers an additional source of supply and its
production can potentially scale up at low cast. Our report here offers an example of this
expression platform serving the cheese production industry. We show here that active
bovine chymosin can be expressed in tobacco plants with a yield of 83.5 mg/kg fresh
weight (approximately 0.52% TSP). This level is similar to the previous report by
Willmitzer et al. [34], who also expressed chymosin in tobacco leaves, but is much
lower than that reported in flex seeds and oilseed rape by van Rooijen et al. [35].
One way to improve upon the expression levels of chymosin reported here would be to
use a codon-optimized gene, as genes from different species may have a particular
genetic codon usage bias [37], and this may crucially impact on the accumulation of
heterogenic proteins in plant. Codon-optimized genes according to the codon usage bias
of target host species [38] guarantee the translation efficiency. Van Rooijen et al. [35]
claimed that the optimized bovine prochymosin gene is suitable to expression in at least
22 plant species listed in the patent, but they only showed the details in flax and oilseed
rape. Indeed, this helped to increase the percentage of chymosin in the total seed protein
of flax (up to around 2.6% TPS) and oilseed rape (average of 4.43% TPS). In the
present report, the DNA sequence of the bovine preprochymosin is taken from a calf
without optimization, which could be one of the reasons leading to a lower expression
level; thus, codon optimizing should be added to further studies.
In addition, there are potentials to modify the transcription of the target gene to improve
the expression of recombinant proteins in plants [39], and the tissue-specific promoters
would be an example by means of modulating the transcription of preprochymosin gene
[34,35]. One report did not seem to have a higher accumulation of chymosin in leaves
(only up to 0.5% TSP) [34], while another report worked well in seeds [35]. In our case,
a constitute promoter CaMV 35S was used to ensure the expression in all tissues, as we
expected to harvest the whole plant for protein extraction. It might be better to use an
inducible, non-tissue-specific promoter, as it may reduce the adverse effects on plant
growth.
Stability of foreign proteins expressed in plants could also be a critical issue for their
accumulation. Multiple options could be adopted as solutions [40,41,42]. Secretory
expression or the sorting of proteins to organelles have been applied to partition
heterologous proteins from cytoplasm, where various house-keeping proteases are
present, and hence to reduce the degradation risk of the target proteins. Numerous
studies have demonstrated that heterologous proteins are well protected in apoplast
[43,44,45]. We retained the secretory peptide of the prochymosin in this study with the
expectation to have the protein secreted to the intercellular space. On the other hand,
whether sorting chymosin to various organelles could be advantageous over
intercellular space should be investigated; therefore, constructs having chymosin ORF
fused with organelle-specific transit peptides should be tested in the future.
Chymosin self-activates at pH between 4 and 5 [46], but the pH of plant cytoplasm is
slightly alkaline [47]. In order to obtain active chymosin, the enzyme should be stored
in a location of lower pH value. Vacuole and intercellular spaces are ideal choices [48].
According to our results, the chymosin extracted from tobacco leaves exhibited milk-
clotting activity, which implies that the enzyme had been activated in the plant tissue, as
the pH value of crude extract and milk are approximately 7.0 and 6.5 respectively,
which is outside the range of 4.0 to 5.0 for self-activation, providing indirect evidence
that the recombinant chymosin has been correctly transited to the intercellular space
accordingly. Moreover, the presence of active chymosin added to the significance of
this work: The crude extract could be directly applied to milk for cheese processing
without protein purification, and this might reduce the production cost.
 4. Materials and Methods
4.1. Vector Construction
The expression vector harboring the preprochymosin gene from bovine (Bostaurus
domesticus) is a derivative of the binary transformation vector pCAMBIA3301 (the
glufosinate resistant version of the binary vector pCAMBIA-1301, accession No.
AF234297) and was constructed by replacing the GUS gene by the bovine
preprochymosin gene. Basically, primers cymF (5′-
TATAGATCTTCGACCTCGAGATGAGGTGTCTCGTGGTGCTACTTGC-3′) and
cymR (5′-
GGAGGCCTGGATCGACTAGTGGTGGTGGTGGTGGTGGATGGCTTTG-3′) were
used to amplify the bovine preprochymosin gene using plasmid pGEM-T-CYM (kindly
provided by Professor Zhen-Nai Yang) as a template. The product was subsequently
inserted into the backbone of pCAMBIA3301 digested by Nco I and Bst EII (New
England Biolabs, Beijing, China) to create the final expression vector, namely
p33cym11, by the in-fusion method [49]. The vector was verified by sequencing and
then transferred to the Agrobacterium tumefaciens strain EHA105 via a freeze/thaw
method [50].

4.2. Tobacco Transformation and Generation of Transgenic Tobacco

Tobacco (Nicotiana tabacum cv. Petite Havana SRI) seeds were surface sterilized and
sown on Murashige and Skoog (MS) medium [51] in the culture room at 25 °C with a
16 h/8 h light-dark photoperiod. The sterile leaves were collected as explants for
transformation after one month. The vector was transferred into tobacco leaf discs
via Agrobacterium-mediated transformation. After co-culturing for 3 days, the leaf discs
were transferred to rest media (MS medium plus 0.2 mg/L α-naphthylacetic acid, 2
mg/L 6-benzylaminopurine and 500 mg/L carbenicillin) to inhibit the growth
of Agrobacteria for 7 days and then removed to selection medium (rest media plus 2
mg/L glufosinate) for regeneration. The selection media were renewed every 15 days
until the resistant shoots were obtained. The shoots were transferred to a rooting
medium (MS medium supplemented with 2 mg/L glufosinate) for rooting. Leaves from
resistant plants were used to perform molecular analyses, and the transgenic plants
confirmed with Southern hybridization were transferred to soil and cultured in a green
house. During the cultivation, the plants were screen again by wiping leaves with 2
mg/L of glufosinate solution, and the uninjured plants were maintained to grow until
seeds were collected.
4.3. Molecular Analysis
4.3.1. PCR and Southern Blotting Analysis
The genomic DNA were extracted from resistant regenerated plants and used as
templates to carry out PCR reaction to confirm the existence of the preprochymosin
gene in the tobacco genome; DNA from the wild-type plant served as a negative
control. The primers were cymdF (5′-CGTGCCCCTGACCAACTACCTG-3′) and
cymdR (5′-CGGAGGGGGTCAGTGGGTACAT-3′). The PCR-positive plants were
further analyzed via Southern blotting: 20 μg of genomic DNA was digested by Bam HI
and Hind III, respectively, and applied to a 0.8% (w/v) agarose gel for electrophoresing.
The electrophoresis was run for 8 h to separate fragments at a current of 5 V/cm in the
TAE buffer. DNAs were then transferred to a positive-charged Hybond-N+nylon
membrane (Roche, Shanghai, China) and fixed by UV cross-linking. The DNA-fixed
membrane was hybridized at 42 °C for 16 h with the digoxigenin (DIG)-labeled Cym
probe in a hybridization oven, and the immune-link detection procedure and
visualization was performed according to the manufacturer’s manual for the DIG High
Prime DNA Labeling and Detection Starter Kit I (Roche, Shanghai, China).
4.3.2. RT-PCR Analyses
Total RNA was extracted from potential transformants to perform RT-PCR analyses.
Two microgram of total RNA from each plant was used as a template for reverse
transcription reactions to synthesize cDNA, with the RNA from wild-type plant used as
a negative control. The cDNA were used as template to perform PCR analyses with the
primer cymdF and cymdR, under the same condition as the PCR analyses. The
endogenous 5S rRNA was chosen to quantify the initial RNA amount.
4.4. Immunoblotting and ELISA Analyses
Mature leaves of T1 plants were collected to extract total soluble protein (TSP).
Approximately 200 mg of leaf tissue was ground into a fine powder in liquid nitrogen,
and 400 μL of PBS (pH7.4) buffer was added to the powder and mixed vigorously for 5
min; the TSP was in the supernatant after centrifuging and then quantified by the
Bradford method [52]. For each sample, 50 μg of TSP were separated via sodium
dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) on a 12% separating
gel. The blotting was carried out followed the standard protocol [53]. The primary
antibody against bovine chymosin was produced by us from rabbit and anti-rabbit
secondary antibody, conjugated with horse radish peroxidase purchased from Sino
Biological Inc. (Beijing, China). Blots were visualized on an X-ray film by an enhanced
chemiluminescence (ECL) method. The same TSP was used to carry out the ELISA
analysis with the same primary antibody against bovine chymosin, following a protocol
previously described [54]. The TSP from the wild-type plant was used as a negative
control for both of the assays above, and the chymosin expressed from E. coli as well as
commercial bovine chymosin (TCI, Shanghai, China) isolated from calf rumen were
used as positive controls for the immunoblotting.
4.5. Bioactivity Detection of Recombinant Bovine Chymosin
A milk coagulation assay was used to detect the bioactivity of the recombinant bovine
chymosin from tobacco by a method described previously [55]. Briefly, 400 μL of fresh
milk, 55 μL of 100 mM CaCl2, and 50 μL of crude TSP were added to 1.5-mL tubes and
well mixed; then, the tubes were incubated in a shaker at 35 °C at a speed of 70 rpm.
Commercial bovine chymosin (TCI, Shanghai, China) isolated from calf rumen (0.1
mg/mL), the TSP from the wild-type plant, and PBS buffer were used as positive,
negative, and blank controls, respectively.

Acknowledgments
We thank Professor Zhen-Nai Yang from the Institute of Agro-food Technology, Jilin
Academy of Agricultural Sciences (now known as the School of Food and Chemical
Engineering, Beijing Technology and Business University) for providing the vector
with bovine preprochymosin gene. We also thank Professor Peter J. Nixon and Dr. Jian-
Feng Yu from Department of Life Sciences, Imperial College London for revising the
manuscript. This work is financial supported by Innovation Project “Plant Bioreactor
Research” from the Jilin Provincial Science and Technology Department
(No.C52110502).
Author Contributions
Zheng-Yi Wei, Feng Lin, and Shao-Chen Xing conceived and designed the
experiments; Zheng-Yi Wei, Yu-Ying Zhang, Yun-Peng Wang, and Nuo Xu performed
the experiments; Ming-Xia Fan and Xiao-Fang Zhong analyzed the data; Zheng-Yi
Wei, Feng Lin, and Shao-Chen Xing wrote the paper.

Conflicts of Interest
The authors declare no conflict of interest.
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Int. J. Mol. Sci. 2016, 17 (5), 624; doi: 10.3390 / ijms17050624

Artikel

Produksi Bioactive Recombinant Bovine Chymosin di Tembakau Tanaman

Zheng-Yi Wei 1,2, Yu-Ying Zhang 2,3, Yun-Peng Wang 2, Fan Ming-Xia 1, Xiao-Fang
Zhong 2, Nuo Xu 4, Feng Lin 1, * dan Shao-Chen Xing 2, *

Agronomi College, Universitas Pertanian Shenyang, No. 120 Dongling Rd., Shenhe
Distirct, Shenyang 110866, Cina

Laboratorium Bioreaktor Tanaman dan Rekayasa Genetika, Laboratorium Utama

Provinsi Jilin, Bioteknologi Pertanian, Institut Penelitian Bioteknologi, Akademi Ilmu
Pengetahuan Jilin, No. 1363 Shengtai St., Changchun 130033, China

State Key Labortory of Agrobiotechnology, Universitas Pertanian China, No 2 West

Yuanmingyuan Rd., Beijing 100094, Cina

Zona Pendidikan Tinggi Chashan, Universitas Wenzhou, Wenzhou 325035, Cina

Korespondensi: Tel .: + 86-24-8848-7163 (F.L.); + 86-431-8706-3121 (S.-C.X.); Faks:

+ 86-24-8848-7161 (F.L.); + 86-431-8706-3080 (S.-C.X.)

Penyunting Akademis: Marcello Iriti

Diterima: 24 Februari 2016 / Diterima: 14 April 2016 / Dipublikasikan: 28 April 2016

Abstrak
: Chymosin (juga dikenal sebagai rennin) memainkan peran penting dalam koagulasi
susu dalam industri keju. Chymosin secara tradisional diekstraksi dari rumen betis dan
berbiaya tinggi. Di sini, kami menyajikan metode alternatif untuk memproduksi bovine
chymosin dari tanaman tembakau transgenik. Gen CYM, yang mengkodekan
preprochymosin dari bovine, dimasukkan ke dalam genom nuklir tembakau di bawah
kendali promotor mouliflower 35S viral. Integrasi dan transkripsi gen asing
dikonfirmasi dengan analisis Southern blotting dan reverse transcription PCR (RT-
PCR), masing-masing. Analisis imunoblot dilakukan untuk menunjukkan ekspresi
chymosin, dan tingkat ekspresi diukur dengan uji enzyme-linked immunosorbent
(ELISA). Hasil penelitian menunjukkan chymosin bovin rekombinan berhasil
diekspresikan pada tingkat rata-rata 83,5 ng / g berat segar, yaitu 0,52% dari total
protein terlarut. Chymosin yang berasal dari tembakau menunjukkan bioaktifitas
koagulasi susu asli yang sama sebagai produk komersial yang diekstrak dari rumen sapi.

Kata kunci:

chymosin; rennin; tembakau; koagulasi susu; transformasi

1. Perkenalan

Chymosin, lebih dikenal sebagai rennin, adalah enzim industri kunci yang digunakan
untuk memproduksi keju, makanan yang secara historis penting di negara-negara Barat
dan salah satu yang meningkatkan permintaan di negara-negara Timur seperti Cina.
Keju dibedakan dengan rasa dan / atau teksturnya yang bergantung pada komposisi dan
chymosin yang digunakan dalam pengolahan keju untuk mengentalkan susu [1,2,3,4].
Enzim dengan pembekuan tinggi untuk rasio aktivitas proteolitik (nilai C / P) lebih
disukai karena aktivitas proteolitik tinggi akan menyebabkan rasa dan / atau tekstur
yang lebih buruk. Karena bovine chymosin memiliki nilai C / P yang tinggi, sangat
cocok untuk produksi keju [5]. Ini adalah alasan utama mengapa bovine chymosin
masih banyak digunakan dan sering ditambahkan ke sumber-sumber lain dari chymosin
seperti mikroorganisme, tumbuhan, dan hewan lainnya [6,7,8,9,10,11,12,13].

Mengingat produksi chymosin dari rumen betis terbatas, metode alternatif diperlukan
untuk menghasilkan chymosin sapi yang cukup untuk memenuhi permintaan global
yang meningkat. CDNA dari gen yang mengkode chymosin sapi diklon dan dianalisa
pada awal tahun 1980 [14,15], dan, sejak itu, banyak upaya telah dilakukan untuk
mengekspresikan chymosin rekombinan dalam berbagai jenis organisme heterolog.
Keberhasilan ekspresi telah dicapai pada banyak spesies seperti Escherichia coli [16],
spesies Bacillus [17], spesies Aspergillus [18,19,20,21], dan ragi [22,23,24,25].
Kymosins yang dikodekan oleh hewan sekarang membentuk lebih dari 70% dari pasar
chymosin global [26] dengan 80% fermentasi chymosin yang diproduksi sebagai
protein rekombinan dalam mikroorganisme [27], dengan protein yang diproduksi di
Aspergillus dan ragi yang paling banyak digunakan.

Banyak jenis protein termasuk vaksin, antibodi, protein terapeutik, dan enzim industri
telah berhasil diekspresikan menggunakan sistem ekspresi tanaman baik pada tanaman
utuh dan dalam kultur sel tanaman [28,29]. Dibandingkan dengan mikroorganisme,
ekspresi protein asing pada tanaman memiliki sejumlah keunggulan termasuk keamanan
yang unggul dan kapasitas peningkatan, kualitas produk yang lebih baik, lipat protein
yang lebih akurat dan modifikasi pasca-translasi. Yang paling penting, ekspresi pada
tumbuhan menawarkan solusi berbiaya rendah untuk produksi dan penyimpanan
[30,31,32]. Sistem ekspresi tanaman dapat dibagi menjadi dua kategori: stabil dan
sementara; dengan ekspresi stabil yang dicapai baik oleh transformasi nukleus atau
plastid, tergantung pada situasinya.

Agak mengherankan, mengingat pentingnya sebagai enzim industri dan potensinya

sebagai enzim terapeutik untuk beberapa perawatan penyakit [33], ada upaya terbatas
untuk mengekspresikan chymosin pada tanaman. Willmitzer dkk. pertama melaporkan
ekspresi chymosin rekombinan dalam daun tembakau [34]. Contoh kedua dilaporkan
oleh van Rooijen et al., Yang mengembangkan sistem ekspresi benih untuk mencapai
tingkat ekspresi yang lebih tinggi [35]. Dalam makalah ini, kami melaporkan ekspresi
chymosin sapi dalam tanaman tembakau transgenik. Tingkat ekspresi bervariasi antara
tanaman yang berbeda, tetapi hasil tertinggi dari chymosin ditentukan menjadi 83,5 ng /
g berat segar (atau 0,52% dari total protein terlarut (TSP)). Chymosin yang berasal dari
tumbuhan masih aktif dan mampu membekukan susu. Studi ini menawarkan pendekatan
alternatif untuk memproduksi chymosin untuk produksi keju.

Hasil2.1. Konstruksi Vektor dan Transformasi Tembakau Vektor ekspresi bovine

preprochymosin, p33cym11, telah berhasil dibangun. Pada Gambar 1A, gen
preprochymosin dikendalikan oleh promotor virus cauliflower 35S (CaMV 35S) dan
terminator gen nopalin sintase (Nos). Dalam vektor, gen acetyltransferase
phosphinothricin, memberikan resistensi terhadap glufosinate, digunakan sebagai
penanda yang dipilih dalam tembakau. Gambar 1. Generasi tanaman transgenik. (A)
Sisipan T-DNA pada vektor ekspresi bovine preprochymosin (CYM) p33cym11. LB
dan RB mewakili batas kiri dan kanan wilayah T-DNA; PPTR merepresentasikan gen
pengkode ketahanan-glufosinate; CaMV 35S dan 35S polyA mewakili promotor dan
terminator gen virus mosaik 35 kembang kol masing-masing; Lac Zα mengkodekan α-
fragmen dari enzim β-galactosidase; Nos polyA mewakili terminator dari gen sintase
nopalin; (B) Tunas yang tahan diregenerasikan pada media seleksi; (C) Tunas yang
tahan membasahi media rooting; (D) Tanaman ditanam di rumah kaca. Setelah menyeka
larutan glufosinat pada bagian-bagian daun yang dilingkari, tidak ada lesi nekrotik yang
terdeteksi untuk tanaman transgenik (kiri) tetapi gejala yang jelas diamati pada tipe liar
(kanan). Batang skala = 1 cm. Tumbuhan transgenik yang mengandung gen
preproymosin diperoleh melalui transformasi disk daun Agrobacterium-mediated.
Tanaman dihasilkan pada media, ditambah 2 mg / L glufosinate sebagai reagen yang
dapat dipilih. Tanaman transgenik menunjukkan resistensi glufosinate dan menunjukkan
fenotip WT normal (Gambar 1B – D) .

2.2. Analisis Molekul DNA genomik diekstraksi dari tanaman tahan glufosinate untuk
melakukan analisis molekuler. Analisis PCR, dilakukan sebagai layar awal untuk
mengkonfirmasi keberadaan gen preprochymosin, CYM, mengungkapkan bahwa
sebagian besar tanaman yang resisten positif (Gambar 2A). Integrasi gen
preprochymosin selanjutnya dikonfirmasi dengan Southern blotting. Hasilnya
menunjukkan bahwa semua tanaman yang diuji bersifat transgenik tetapi mengandung
salinan gen asing yang berbeda (Gambar 2B). Ada satu salinan di baris 11J dan dua atau
lebih salinan di empat baris lainnya. Hasil RT-PCR menunjukkan bahwa CYMgene
ditranskripsikan di semua tanaman yang diuji (Gambar 2C), tetapi tampaknya tidak ada
hubungan antara tingkat transkripsi dan nomor salinan yang dimasukkan. Baris 11L,
misalnya, memiliki dua salinan gen target, tetapi menunjukkan akumulasi mRNA
terendah, sementara garis 11J menyimpan salinan tunggal diamati dengan akumulasi
yang jauh lebih tinggi. Gambar 2. Analisis molekuler tanaman transgenik. (A) analisis
PCR dari tanaman regenerasi yang tahan. M, DNA ladder (Transgene, Beijing, China,
cat. No.BM301); +, vektor p33cym11 sebagai kontrol positif; -, H2O sebagai kontrol
kosong; CK, tanaman jenis liar; Jalur 1-10, tanaman tahan; (B) Analisis Southern
Blotting menggunakan fragmen CYM sebagai probe untuk hibridisasi dengan DNA
dicerna oleh Bam HI dan Hind III. M, DNA ladder (lambda DNA yang dicerna oleh Sty
I); +, vektor p33cym11 sebagai kontrol positif; CK, tanaman jenis liar; 11A, 11C, 11I,
11J, dan 11L adalah garis transgenik yang berbeda; (C) Analisis transkripsi gen CYM
pada tanaman transgenik. Cym menunjukkan band-band khusus untuk gen CYM, 5S
rRNA menunjukkan jumlah RNA yang digunakan dalam analisis. 11I, 11A, 11J, 11L,
dan 11C adalah garis transgenik yang berbeda; +, vektor p33cym11 sebagai kontrol
positif; CK, tanaman tipe liar

.2.3. Ekspresi Chymosin Bovin Rekombinan dan Deteksi Aktivitas Immunoblotting

mengkonfirmasi keberhasilan ekspresi kimosin sapi di TSP yang diekstrak dari tanaman
(Gambar 3A). Analisis kuantitatif oleh ELISA menunjukkan bahwa tingkat ekspresi
bervariasi dari 18,1 hingga 83,5 ng / g berat segar, yang berarti 0,18% hingga 0,52%
TSP (Gambar 3B). Gambar 3. Ekspresi chymosin bovin rekombinan di tanaman
tembakau transgenik. (A) Analisis Immunoblotting; (B) Hasil dari rekombinan bovin
chymosin dalam tanaman transgenik. Chymosin, CK, dan rProchymosin mewakili
chymosin komersial yang diisolasi dari betis, TSP mentah dari tanaman wild type dan
prochymosin rekombinan dengan 6 × tag-Nya dari E. coli; 11A, 11C, 11I, 11J, dan 11L
mewakili jalur tanaman transgenik individu yang berbeda atau TSP mentah dari mereka.
Data hasil dikumpulkan dari tiga duplikat independen untuk setiap jalur. Pengujian
bioaktivitas dilakukan untuk menguji apakah tanaman tersebut berasal masih berfungsi.
Dalam pengujian ini, waktu yang dibutuhkan untuk susu segar akan dibekukan oleh
TSP tanaman dinilai. Semua tanaman yang mengekspresikan chymosin menunjukkan
bioaktivitas alami. Sebagai tingkat ekspresi bervariasi dalam tanaman transgenik yang
berbeda, chymosin mentah menunjukkan tingkat bioaktivitas yang berbeda, pada waktu
pembekuan susu mulai dari 20,5 hingga 46,3 menit (Gambar 4). Menariknya, akumulasi
mRNA mungkin berkorelasi dengan hasil protein. Gambar 4. Tes pembekuan susu
untuk chymosin bovin rekombinan. Status pembekuan susu (A) dan waktu yang
dikonsumsi (B) diperlihatkan. Chymosin, PBS, dan CK mewakili chymosin komersial
dari betis, buffer PBS, dan TSP mentah dari tanaman wild type masing-masing; 11A,
11C, 11I, 11J, dan 11L mewakili TSP mentah dari tanaman transgenik. Data waktu
pembekuan dikumpulkan dari tiga pengulangan independen.
PEMBAHASAN Meskipun chymosin sapi telah dinyatakan dalam banyak
mikroorganisme, produksi masih belum dapat memenuhi permintaan pasar [36].
Ekspresi chymosin pada tanaman mungkin membantu mengatasi kekurangan ini karena
pabrik GM menawarkan sumber suplai tambahan dan produksinya dapat berpotensi
meningkat pada pemeran rendah. Laporan kami di sini menawarkan contoh platform
ekspresi ini melayani industri produksi keju. Kami menunjukkan di sini bahwa aktif
chymosin sapi dapat dinyatakan dalam tanaman tembakau dengan hasil 83,5 mg / kg
berat segar (sekitar 0,52% TSP). Tingkat ini mirip dengan laporan sebelumnya oleh
Willmitzer et al. [34], yang juga mengungkapkan chymosin dalam daun tembakau,
tetapi jauh lebih rendah daripada yang dilaporkan pada biji fleksibel dan pemerkosaan
minyak oleh van Rooijen et al. [35]. Salah satu cara untuk meningkatkan tingkat
ekspresi chymosin yang dilaporkan di sini adalah menggunakan gen yang dioptimalkan
kodon, karena gen dari spesies yang berbeda mungkin memiliki bias penggunaan kodon
genetik tertentu [37], dan ini dapat sangat berdampak pada akumulasi protein heterogen
di tanaman. Gen yang dioptimalkan oleh codon sesuai dengan bias penggunaan kodon
dari spesies inang target [38] menjamin efisiensi penerjemahan. Van Rooijen dkk. [35]
menyatakan bahwa gen prochymosin sapi yang dioptimalkan cocok untuk berekspresi
di setidaknya 22 spesies tanaman yang terdaftar di paten, tetapi mereka hanya
menunjukkan rincian dalam rami dan biji rami. Memang, ini membantu meningkatkan
persentase chymosin dalam total protein biji rami (hingga sekitar 2,6% TPS) dan
pemerkosaan biji minyak (rata-rata 4,43% TPS). Dalam laporan ini, urutan DNA
preprochymosin sapi diambil dari anak sapi tanpa optimasi, yang bisa menjadi salah
satu alasan yang mengarah ke tingkat ekspresi yang lebih rendah; dengan demikian,
mengoptimalkan kodon harus ditambahkan ke studi lebih lanjut. Selain itu, ada potensi
untuk memodifikasi transkripsi gen target untuk meningkatkan ekspresi protein
rekombinan pada tanaman [39], dan promotor spesifik jaringan akan menjadi contoh
dengan cara modulasi transkripsi gen preprochymosin [34,35]. Satu laporan tampaknya
tidak memiliki akumulasi lebih tinggi dari chymosin dalam daun (hanya hingga 0,5%
TSP) [34], sementara laporan lain bekerja dengan baik dalam biji [35]. Dalam kasus
kami, promotor CaMV 35S digunakan untuk memastikan ekspresi di semua jaringan,
seperti yang kami harapkan untuk memanen seluruh tanaman untuk ekstraksi protein.
Mungkin lebih baik menggunakan promotor non-jaringan spesifik yang dapat diinduksi,
karena dapat mengurangi efek buruk pada pertumbuhan tanaman. Stabilitas protein
asing yang diekspresikan pada tanaman juga bisa menjadi masalah kritis untuk
akumulasi mereka. Berbagai pilihan dapat diadopsi sebagai solusi [40,41,42]. Ekspresi
sekretori atau penyortiran protein ke organel telah diterapkan untuk mempartisi protein
heterolog dari sitoplasma, di mana berbagai protease pemeliharaan rumah hadir, dan
karenanya untuk mengurangi risiko degradasi protein target. Sejumlah penelitian telah
menunjukkan bahwa protein heterolog terlindungi dengan baik dalam apoplast
[43,44,45]. Kami mempertahankan peptida sekretorik prochymosin dalam penelitian ini
dengan harapan untuk memiliki protein yang disekresikan ke ruang antar sel. Di sisi
lain, apakah penyortiran chymosin ke berbagai organel dapat menguntungkan daripada
ruang interseluler harus diselidiki; oleh karena itu, konstruk memiliki chymosin ORF
yang menyatu dengan peptida transit spesifik-organel harus diuji di masa depan.
Chymosin self-activates pada pH antara 4 dan 5 [46], tetapi pH sitoplasma tumbuhan
sedikit bersifat basa [47]. Untuk mendapatkan chymosin aktif, enzim harus disimpan di
lokasi dengan nilai pH yang lebih rendah. Vakuole dan ruang interseluler adalah pilihan
ideal [48]. Menurut hasil kami, chymosin yang diekstrak dari daun tembakau
menunjukkan aktivitas pembekuan darah, yang menyiratkan bahwa enzim telah
diaktifkan di jaringan tanaman, karena nilai pH ekstrak kasar dan susu masing-masing
sekitar 7,0 dan 6,5, yang berada di luar kisaran 4,0-5,0 untuk aktivasi diri, memberikan
bukti tidak langsung bahwa chymosin rekombinan telah benar transit ke ruang antar
selaras. Selain itu, kehadiran aktif chymosin ditambahkan ke signifikansi dari pekerjaan
ini: Ekstrak mentah dapat langsung diterapkan pada susu untuk pengolahan keju tanpa
pemurnian protein, dan ini dapat mengurangi biaya produksi. Bahan dan Metode4.1.
Konstruksi Vektor Vektor ekspresi yang mengandung gen preprochymosin dari bovine
(Bostaurus domesticus) adalah turunan dari vektor transformasi biner pCAMBIA3301
(versi tahan glufosinate dari vektor biner pCAMBIA-1301, aksesi No. AF234297) dan
dikonstruksi dengan mengganti gen GUS oleh gen preprochymosin bovin. Pada
dasarnya, primer cymF (5′-
TATAGATCTTCGACCTCGAGATGAGGTGTCTCGTGGTGCTACTTGC-3 ′) dan
cymR (5′-GGAGGCCTGGATCGACTAGTGGTGGTGGTGGTGGATGGCTTTG-3 ′)
digunakan untuk memperkuat gen preprochymosin bovine menggunakan plasmid
pGEM-T-CYM (disediakan oleh Profesor Zhen-Nai Yang) sebagai sebuah template.
Produk tersebut kemudian dimasukkan ke dalam tulang punggung pCAMBIA3301 yang
dicerna oleh Nco I dan Bst EII (New England Biolabs, Beijing, China) untuk
menciptakan vektor ekspresi akhir, yaitu p33cym11, dengan metode in-fusion [49].
Vektor diverifikasi oleh sekuensing dan kemudian ditransfer ke strain Agrobacterium
tumefaciens EHA105 melalui metode freeze / thaw [50]

.4.2. Transformasi Tembakau dan Generasi Benih Tembakau TransgenikTobacco

(Nicotiana tabacum cv. Petite Havana SRI) telah disterilkan dan ditaburkan pada media
Murashige dan Skoog (MS) [51] di ruang budaya pada suhu 25 ° C dengan lampu 16
jam / 8 jam fotoperiode gelap. Daun steril dikumpulkan sebagai eksplan untuk
transformasi setelah satu bulan. Vektor dipindahkan ke cakram daun tembakau melalui
transformasi yang dimediasi Agrobacterium. Setelah ko-kultur selama 3 hari, cakram
daun dipindahkan ke media istirahat (media MS ditambah 0,2 mg / L α-naphthylacetic
acid, 2 mg / L 6-benzylaminopurine dan 500 mg / L carbenicillin) untuk menghambat
pertumbuhan Agrobacteria untuk 7 hari dan kemudian dipindahkan ke media seleksi
(sisa media ditambah 2 mg / L glufosinate) untuk regenerasi. Media seleksi diperbarui
setiap 15 hari sampai tunas resisten diperoleh. Tunas dipindahkan ke media rooting
(media MS yang dilengkapi dengan 2 mg / L glufosinate) untuk rooting. Daun dari
tanaman tahan digunakan untuk melakukan analisis molekuler, dan tanaman transgenik
dikonfirmasi dengan hibridisasi Selatan dipindahkan ke tanah dan dibudidayakan di
rumah hijau. Selama budidaya, tanaman disaring lagi dengan mengelap daun dengan 2
mg / L larutan glufosinat, dan tanaman yang tidak terluka dipertahankan untuk tumbuh
sampai biji dikumpulkan.

4.3. Analisis Molekuler

4.3.1. PCR dan Southern Blotting Analysis DNA genom diekstraksi dari tanaman yang
diregenerasi dan digunakan sebagai templat untuk melakukan reaksi PCR untuk
mengkonfirmasi keberadaan gen preprochososin dalam genom tembakau; DNA dari
tumbuhan liar berfungsi sebagai kontrol negatif. Primer adalah cymdF (5′-
CGTGCCCCTGACCAACTACCTG-3 ′) dan cymdR (5′-
CGGAGGGGGTCAGTGGGTACAT-3 ′). Tanaman PCR-positif dianalisis lebih lanjut
melalui Southern blotting: 20 μg DNA genomik dicerna oleh Bam HI dan Hind III,
masing-masing, dan diterapkan pada gel agarosa 0,8% (w / v) untuk elektroforesis.
Elektroforesis dijalankan selama 8 jam untuk memisahkan fragmen pada arus 5 V / cm
dalam buffer TAE. DNA kemudian dipindahkan ke membran nilon Hybond-N +
bermuatan positif (Roche, Shanghai, China) dan diperbaiki dengan UV cross-linking.
Membran DNA-tetap hibridisasi pada 42 ° C selama 16 jam dengan digoxigenin (DIG) -
label Cym probe dalam oven hibridisasi, dan prosedur deteksi imun-link dan visualisasi
dilakukan sesuai dengan manual pabrik untuk DIG High Prime Pelabelan DNA dan
Deteksi Starter Kit I (Roche, Shanghai, Cina) .

4.3.2. Analisis RT-PCRTotal RNA diekstrak dari transforman potensial untuk

melakukan analisis RT-PCR. Dua mikrogram total RNA dari masing-masing pabrik
digunakan sebagai template untuk reaksi transkripsi terbalik untuk mensintesis cDNA,
dengan RNA dari tanaman wild type yang digunakan sebagai kontrol negatif. CDNA
digunakan sebagai template untuk melakukan analisis PCR dengan cymdF primer dan
cymdR, di bawah kondisi yang sama seperti analisis PCR. 5S rRNA endogen dipilih
untuk mengukur jumlah RNA awal.

4.4. Immunoblotting dan ELISA Analyses

Daun daun tanaman T1 dikumpulkan untuk mengekstrak total protein terlarut (TSP).
Sekitar 200 mg jaringan daun digiling menjadi bubuk halus dalam nitrogen cair, dan
400 μL buffer PBS (pH7.4) ditambahkan ke bubuk dan dicampur dengan kuat selama 5
menit; TSP berada di supernatan setelah sentrifugasi dan kemudian dikuantifikasi
dengan metode Bradford [52]. Untuk setiap sampel, 50 μg TSP dipisahkan melalui
elektroforesis gel natrium dodecyl sulfate-polyacrylamide (SDS-PAGE) pada gel
pemisah 12%. Blotting dilakukan mengikuti protokol standar [53]. Antibodi utama
terhadap chymosin sapi diproduksi oleh kami dari kelinci dan antibodi sekunder anti-
kelinci, terkonjugasi dengan peroksidase kuda yang dibeli dari Sino Biological Inc.
(Beijing, China). Blot divisualisasikan pada film X-ray dengan metode
chemiluminescence (ECL) yang ditingkatkan. TSP yang sama digunakan untuk
melakukan analisis ELISA dengan antibodi primer yang sama terhadap bovine
chymosin, mengikuti protokol yang dijelaskan sebelumnya [54]. TSP dari tanaman wild
type digunakan sebagai kontrol negatif untuk kedua tes di atas, dan chymosin yang
diekspresikan dari E. coli serta chymosin sapi komersial (TCI, Shanghai, China) yang
diisolasi dari rumen betis digunakan sebagai positif. kontrol untuk imunoblotting.

4.5. Deteksi Bioaktifitas dari Rekombinan Bovine Chymosin

Uji koagulasi susu digunakan untuk mendeteksi bioaktifitas chymosin bovin

rekombinan dari tembakau dengan metode yang dijelaskan sebelumnya [55]. Secara
singkat, 400 μL susu segar, 55 μL 100 mM CaCl2, dan 50 μL TSP mentah ditambahkan
ke tabung 1,5 mL dan dicampur dengan baik; kemudian, tabung diinkubasi dalam
shaker pada 35 ° C dengan kecepatan 70 rpm. Komersial chymosin sapi (TCI, Shanghai,
Cina) diisolasi dari rumen betis (0,1 mg / mL), TSP dari tanaman liar, dan PBS buffer
digunakan sebagai kontrol positif, negatif, dan kosong, masing-masing.

Ucapan terima kasih

Kami berterima kasih kepada Profesor Zhen-Nai Yang dari Institut Teknologi Agro-
Makanan, Akademi Ilmu Pertanian Jilin (sekarang dikenal sebagai Sekolah Teknik
Pangan dan Kimia, Teknologi Beijing dan Universitas Bisnis) untuk menyediakan
vektor dengan gen bovin preprochymosin. Kami juga berterima kasih kepada Profesor
Peter J. Nixon dan Dr. Jian-Feng Yu dari Departemen Ilmu Hayat, Imperial College
London untuk merevisi naskah. Pekerjaan ini didukung oleh Proyek Inovasi "Penelitian
Bioreaktor Tanaman" dari Departemen Sains dan Teknologi Provinsi Jilin
(No.C52110502).