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Ectopic expression of Recombinant human IGF-1 protein
in Nicotiana benthamiana and Nicotiana tabacum BY-2 cells
Kwangkyun Park , Il-Chul Kim , Dong Ju Lee 1,2
2
2
Basic Science Institute , Department of Biological Sciences , Chonnam National University, Gwangju 61186, South Korea
2
1
Introduction Results
• What is IGF-1?
• RT-PCR & DNA electrophoresis
Human IGF-1 is a hormone mainly secreted RT-PCR was conducted using rhIGF-1 gene-specific primer about transgenic N. benthamiana
from the liver into blood vessels, which is and N. tabacum cv. BY-2 cells homozygote lines. Then, DNA-electrophoresis was
controlled by Growth Hormone (GH) from implemented in 1.2 % agarose gel. As a result, amplified cDNA bands of approximately 500
the pituitary gland. Through blood bp were observed, which means the certainty of plant transformation by A. tumefaciens.
circulation, IGF-1 reaches out to various
tissues, binds to IGF-1 Receptor (IGF-1R) or
Insulin Receptor (IR), and induces cell N. benthamiana BY-2
proliferation and survival, tissue
regeneration, and glucose concentration Line 1 2 Line 1 2 3 6 12
control in blood. Therefore, IGF-1 has an
important role in the body of overall
growth, neuroprotection, tissue repair, bone
formation, and glycemic control. If there is
any defect in GH production, GH action, rhIGF cDNA band
IGF-1 production, or IGF-1 action, the (500 bp) rhIGF cDNA band
normal level of IGF-1 is not sustained and (500 bp)
may cause many related diseases, such as
IGF-1 deficiency, diabetes, chronic renal
insufficiency, and amyotrophic lateral
sclerosis. Especially, primary IGF-1
deficiency is characterized by conspicuous • Western Blot
short stature and reported taking up to Western Blot was conducted in 5-13% discontinuous polyacrylamide gel about the protein
50% of idiopathic short stature children. extractions which were standardized for 100ug of soluble proteins by Bradford Assay. As a
Therefore, human IGF-1 has a high clinical result, rhIGF-1 bands of approximately 32kDa were observed, evaluating the quantification
significance in medicine. of expressed rhIGF-1 amounts.
• What is Molecular pharming? <Yield>
Line (ng)
Molecular pharming is a biotechnological Nb2 95.8
manipulation of plant cells for the sake Nb4 101.4
of the production and utilization of a Nb23 96.8
medically valuable protein. Plant Nb24 99.5
production systems have significantly Nb27 111.9
lower cost than mammalian cell cultures
and are enable performing eukaryotic ~0.11% (1.12 ug/mg) of total soluble protein
PTMs, while not carrying human
pathogens.
N. tabacum BY-2
<Yield>
Hence, this study was designed Line (ng)
considering both the pharmacological Line 2 3 6 2 BY2 168.6
value of human IGF-1 and the strength of KNO (g/L) 0 10 BY3 37
the plant production system. 3 BY6 130.6
IGF-1 (ng) 80 120 160 BY2KNO 0 105.3
3
Methods BY2KNO 10 187.5
3
32 kDa ~0.27% (2.69 ug/mg) of total soluble protein
(rhIGF-1)
• Plant cell transformation by Agrobacterium tumefaciens Ti plasmid
Discussion
Human IGF-1 gene
Insertion
Commercial value is one of the most important parts of the recombinant protein industry,
which is represented by yield, production cost, and activity of the protein.
The yield of recombinant human IGF-1(rhIGF-1) was ~0.11% (1.12 ug/mg total soluble
protein) in N. benthamiana and ~0.27% (2.69 ug/mg total soluble protein) in N. tabacum
BY-2. Both yields are considered conspicuously high when compared to another study [5]
that rhIGF-1 yields reach 113±24 ng/mg in transformed Oryza sativa and 70±13 ng/mg in
Infection
transformed N. tabacum, respetively.
The production cost is estimated relatively very low in molecular pharming. Although,
seemingly, our results indicate better yield in BY-2 cells, the BY-2 cell culture system is likely
to require more expenses due to its media. On the other hand, transgenic N. benthamiana
plant can be cultivated on soil and harvested massively depending on the scale of pharming.
The activity of this plant-derived rhIGF-1 should be estimated for suitability for human or
other mammalian cell cultures. The molecular weight of human IGF-1 is known for 7.6 kDa,
but this rhIGF-1 from plants weighs approximately 32 kDa, which, assumably, is plant-
specifically hyperglycosylated. As an alternative, it may find its application in mammalian
cell culture, for example, cultured meat. To address this hyperglycosylation, a downstream
process may be needed to remove glycans. Moreover, many other studies that used
glycoengineering by gene editing, e.g. knock-out of plant-specific glycosyltransferase,
• Reverse transcription polymerase chain reaction (RT-PCR) • Western blot (WB) knock-in of human β(1,4)galactosyltransferase.
Furthermore, other ways for rhIGF-1 yield boosting are currently considered and being
Signal
Substrate verified. KNO3 rich environment is effective, but the experiment should be conducted
repeatedly and needs to be optimized.
5’ A-A-A-A 3’ mRNA
References
3’ N-N-N-N 5’ rhIGF-1 gene specific primer
Enzyme
Secondary
mRNA
Antibody [1] Introduction of New Recombinant Insulin-like Growth Factor-1 – Current and Future Perspectives a report by Michael B Ranke Professor
cDNA of Paediatrics, and Head, Section of Paediatric Endocrinology, University of Tübingen Children’s Hospital DOI:10.17925/EE.2008.04.00.52 [2]
Converting Insulin-like Growth Factors 1 and 2 into High-Affinity Ligands for Insulin Receptor Isoform A by the Introduction of an
Evolutionarily Divergent Mutation. Biochmistry2018, 57, 2373-2382. DOI: 10.1021/acs.biochem.7b01260 [3] Rozov SM, Permyakova NV,
Primary Deineko EV. Main Strategies of Plant Expression System Glycoengineering for Producing Humanized Recombinant Pharmaceutical Proteins.
Biochemistry (Mosc). 2018 Mar;83(3):215-232. doi: 10.1134/S0006297918030033 [4] Primary IGF-1 Deficiency Is a Frequent Cause of Short
x 2 n Antibody Stature 13-May-2005 2:00 PM EDT, by University of California, Los Angeles (UCLA), Health Sciences [5] Panahi, M., Alli, Z., Cheng, X. et
cDNA
al. Recombinant Protein Expression Plasmids Optimized for Industrial E. coli Fermentation and Plant Systems Produce Biologically Active
Human Insulin-like Growth Factor-1 in Transgenic Rice and Tobacco Plants. Transgenic Res 13, 245–259 (2004).
https://doi.org/10.1023/B:TRAG.0000034619.21613.d0
Protein
Acknowledgement
PVDF membrane
This research was supported by Basic Science Research Program through the National Research Foundation of
Korea(NRF) funded by the Ministry of Education(2021R1I1A1A01053989)
