Transgenic Ginseng Cell Lines That Produce High levels of a Human Lactoferrin

• Abstract
• Abbreviations
• Introduction
• Materials and Methods
• Transformation and expression vector
• Protein extraction and determination of hLf content
• Southern and northern blot analysis
• Western blot analysis
• Results and Discussion
• Transformation and cell line selection
• Molecular analysis of hLf expressing cell lines
• Acknowledgements
• References

Abstract

In order to produce a human lactoferrin (hLf) protein in cultured plant cells, we developed Korean ginseng (Panax ginseng) cell line using an oxidative stress-inducible peroxidase (SWPA2) promoter and characterized the production of human lactoferrin in cultured cells. A construct containing a targeting signal peptide from tobacco endoplasmic reticulum fused to human lactoferrin cDNA under the control of SWPA2 promoter was engineered. Transgenic Korean ginseng cell lines that produced a recombinant hLf protein were successfully generated and confirmed by PCR and Southern blot analyses. Western blot and ELISA analyses showed that hLf protein was synthesized in the transgenic cells. The production of hLf showed a maximal level (up to 3.0 % of total soluble protein) in the stationary phase of callus cultures. These results suggest that the transgenic cell lines in this study will be biotechnologically useful for the commercial production of hLf protein in cell cultures, with no need for purification.

Abbreviations

ELISA:enzyme-linked immunosorbent assay

TSP:total soluble protein

MS:Murashige and Skoog

DAS:days after subculture

Introduction

Lactoferrin is an iron-binding glycoprotein of approximately 80 kDa, which was originally found in milk. Human lactoferrin (hLf) plays significant protective roles in human milk, such as antibacterial [1], antifungal [2], anti-endotoxin [3], and antiviral activities [4]. Recombinant hLf has been produced in fungi [5], yeast [6], and mammalian systems, including cows [7]. However, the animal and fungi production systems require expensive purification processes, and harbor harmful mammalian disease-causing viruses, microbes, fungi and prions of an animal cell origin [8].

Mitra and Zhang [9] first reported the hLf gene expression under the control of the CaMV 35S promoter in cultured tobacco cells, which contained approximately 1.8 % of total soluble protein. However, low levels of hLf have been expressed in tobacco leaves [10], rice [11], and potatoes [12], with 0.3 % of total soluble protein.

Cultured plant cells have become attractive systems for the production of secondary metabolites and recombinant proteins post transformation. The great advantage of plant cell cultures is that recombinant proteins can be produced under certified conditions, with a low cost for large-scale production, but the yields are low compared with stably transformed plants and yeast [13].

Medicinal plant cell cultures have been applied commercially for use in various health teas and foods [14]. Thus, the mass production of plant cells and tissues, through large-scale suspension cultures, can be directly applied to the production of pharmacologically active components and of fresh raw materials. The expression of recombinant hLf in edible tissues, such as banana or ginseng, has the advantage, over other systems, e.g., tobacco, of eliminating the need to purify the transgenic protein from the producing organism prior to feeding. However, there is no report on transgenic plants, or cell lines of medicinal plants, producing useful biomaterials, such as hLf proteins.

Ginseng (Panax ginseng C. A. Meyer) is an herbaceous perennial medicinal plant that is cultivated for its highly valued roots, which provide a source of revitalizing and stimulating agents [15]. Plant regeneration, through somatic embryogenesis, and the development of transgenic ginseng has previously been reported [16], [17]. In this paper, we describe, for the first time, a transgenic ginseng cell line using oxidative stress-inducible peroxidase SWPA2 promoter with high levels of hLf expression.

Materials and Methods

Transformation and expression vector

Cotyledons of dehusked mature zygotic embryos of ginseng (Panax ginseng C. A. Meyer) were excised and cocultured with Agrobacterium tumefaciens harboring the binary vector SWPA2pro::ER-hLf/pCGN1578 (Fig. [1]), for 48 h in MS [18] liquid medium containing 1 mg/l, 2,4-dichlorophenoxyacetic acid (2,4-D). The cultures were rinsed 3 to 4 times in the culture medium, and placed with their adaxial surfaces down on the MS medium, which was supplemented with 1 mg/l 2,4-D, 200 mg/l kanamycin, and 300 mg/l claforan (selection medium). They were cultured at 25 °C in the dark. Individual kanamycin-resistant calli were transferred to fresh selection medium every 3 weeks over 10-month period until the culture was established. Samples for analysis were taken on 20 days after subculture.

Protein extraction and determination of hLf content

Transformed ginseng cells (1 g fresh weight) were homogenized on ice in a mortar with an equal volume of ice-cold extraction buffer (50 mM potassium phosphate, pH 7.0) and centrifuged at 12,000 g and 4 °C for 15 min. An enzyme-linked immunosorbent assay (ELISA) was performed on the protein extracts from the transformed and non-transformed (control) cells. The presence of recombinant hLf was assessed by ELISA, according to the BIOXYTECH® Lactof-EIA^TM (OxiResearch^TM). Each experiment was repeated at least three times.

Southern and northern blot analysis

Genomic DNA was extracted from the ginseng cell lines containing high levels of hLf protein, and digested with EcoRI. The genomic DNA underwent electrophoresis on a 0.8 % agarose gel, were blotted onto a Zeta-Probe GT membrane (Bio-Rad), and hybridized to the 1.0 kb of an hLf DNA-probe. The hybridization was carried out in 0.25 M sodium phosphate (pH 7.2), containing 7 % SDS, at 60 °C. After hybridization, the blot was washed once with 20 mM sodium phosphate (pH 7.2), and 1 % SDS at room temperature for 10 min, then twice with the same solution at 60 °C. For the northern blot analysis, total RNA was extracted with a TRIzol^TM (GIBCO/BRL), following the manufacturer's instructions. Approximately 20 μg of total RNA underwent electrophoresis on a 1 % agarose gel containing 0.67 M formaldehyde, and blotted onto a Zeta membrane (Bio-Rad). Radiolabel probing and hybridization were performed as above.

Western blot analysis

Total soluble protein was extracted from the transformed ginseng cells described above. Protein extracts (20 μg) were separated on a 10 % (w/v) acrylamide gel, with 100 ng of commercially available lactoferrin (Sigma) as a standard. The resolved proteins were transferred to a PVDF nylon membrane (Millipore Co.), and immersed in a blocking solution (1 % BSA and 10 mM Tris-HCl, pH 7.4, and 150 mM NaCl) for 1 hr at 4 °C. The membrane was incubated for overnight in a 1 : 20,000 dilution of a commercially available polyclonal antibody conjugated with peroxidase (Rabbit anti-Human Lactoferrin, BIODESIGN International). After washing five times with TBST buffer (10 mM Tris-HCl, pH 7.4, 150 mM NaCl, and 0.1 % Tween 20), the blot was detected using the ECL plus Western Blotting Detection System (Amersham Pharmacia Biotech UK Limited), following the manufacturer's instructions.

Results and Discussion

Transformation and cell line selection

Cotyledon explants of the zygotic embryos of ginseng were transformed by Agrobacterium tumefaciens carrying the SWPA2pro:ER-hLf/pCGN1578 plasmid, as shown in Fig. [1]. Transformed ginseng calli were selected on MS medium containing 200 mg/l kanamycin. Kanamycin-resistant calli were formed after 8 weeks of culturing on selection medium (Fig. [2] A, B), and maintained in the same medium (Fig. [2] C, D). After subculturing, the kanamycin-resistant callus lines were generated, and the presence of the hLf gene in the ginseng callus lines was detected by PCR (data not shown here). The transformed ginseng calli had a normal appearance, and similar growth characteristics as the non-transformed control cells.

The levels of hLf protein in nineteen independent PCR positive callus lines, 20 days after subculture (DAS), were estimated by an ELISA method. A quantitative analysis revealed that the content of hLf was in the range of 55.9 μg/g FW (fresh weight) (#16) to 155.5 μg/g FW (#18) (Fig. [3] A). The transgenic calli expressed hLf contents ranging from 0.1 % to 3.0 % of the total soluble protein (Fig. [3] B). The expression of hLf in ginseng callus lines, under the control of the SWPA2 promoter, was approximately 2 to 10-fold higher than the amount of hLf generated by the other promoters [9] [10] [11] [12].

The high level of hLf expression may be due to the strong oxidative stress inducible SWPA2 promoter. The SWPA2 promoter is a strong oxidative stress-inducible POD promoter, which was cloned from sweetpotato (Ipomoea batatas) [19]. The SWPA2 promoter was strongly expressed in cultured cells, and highly induced in response to environmental stresses in intact plants. Furthermore, GUS protein in suspension cultures of transgenic tobacco cells, using a SWPA2 promoter, was strongly expressed following the stationary phase of cell growth. Therefore, we anticipate that the SWPA2 promoter will the biotechnologically useful for the development of particular transgenic cell lines engineered to produce key pharmaceutical proteins. The suspension cultures of transgenic ginseng cell lines are being established.

Molecular analysis of hLf expressing cell lines

To confirm the stable integration of the hLf gene into the nuclear genome of host cells, a Southern blot analysis was performed on the six ginseng callus lines (#3, #4, #7, #14, #18, and #19) with high levels of hLf protein, using ³²P-labeled hLf cDNA as a probe (Fig. [4] A). After EcoRI digestion, six callus lines produced single band, indicating that the hLf gene was properly incorporated into the genomic DNA of ginseng callus. The non-transgenic control ginseng callus showed no bands. Actually EcoRI excised the transgene, and hence was not suitable to show integration in different genomic location or possible differences in copy number between the lines. Unfortunately, many restriction enzymes excised the transgene, and a few restriction enzymes would not completely digest genomic DNA.

Total RNA, isolated from the same cell lines (#3, #4, #7, #14, #18, and #19), was analyzed by northern hybridization with an hLf-specific probe to examine the hLf gene expression in the transgenic ginseng cells. The northern blot analysis indicated the presence of hLf transcripts (Fig. [4] B). The hLf gene was highly expressed in four of the transgenic ginseng cell lines, #3, #7, #14, and #18, but was detected at very low levels in the other transgenic cell lines. The hLf gene expression was not detectable in the non-transformed control cells.

The transgenic ginseng cell lines were tested for the expression of the hLf protein by Western blot analysis. The recombinant hLf protein synthesized in the six transgenic callus lines (#3, #4, #7, #14, #18 and #19) is shown in Fig. [4] C. The immunoreactive hLf levels differed between the transgenic calli lines tested. The transgenic ginseng callus lines produced the hLf protein of 80 kDa and 40 kDa, but the extracts from the non-transformed (C) calli did not react with the anti-hLf antibody. Three callus lines produced an hLf protein of 80 kDa only. These results suggested that partial-length hLf resulted from degradation during the process of soluble protein extraction or premature translation termination due to extreme overexpression in cells.

In previous other studies, Mitra & Zhang [9] reported the expression of hLf in tobacco calli, which produced only truncated hLf protein, with a molecular weight of 48 kDa. Recently, a full-length hLf was isolated from transgenic tobacco and potato plants [10], [12]. However, it is not known why a partial-length lactoferrin is produced in tobacco cells. It is possible that the plant-produced partial-length hLf protein because it does not undergo proper folding, and the unfolded part is degraded [9].

In this study, we described the use of ginseng calli as a system for the production of hLf expression. If the transgenic cultured cells of ginseng contain pharmacologically active components, the mass production of cultured ginseng cells could be used to produce raw medicinal materials. Thus, this system could be applied to the industrial production of pharmacologically active components.

Acknowledgements

This research was supported by a grant (#PF0330602-02) from Plant Diversity Research Center of 21st Century Frontier Research Program funded by Ministry of Science and Technology of Korean government.

References

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  PubMedSearch in Google Scholar
• 18 Murashige T, Skoog F. A revised medium for rapid growth and bioassays with tobacco tissue culture.  Physiol Plant. 1962;  15 473-97
  CrossrefPubMedSearch in Google Scholar
• 19 Kim K Y, Kwon S Y, Lee H S, Hur Y, Bang J W, Kwak S S. A novel oxidative stress-inducible peroxidase promoter from sweet potato: molecular cloning and characterization in transgenic tobacco plants and cultured cells.  Plant Mol Biol. 2003;  51 831-8
  CrossrefPubMedSearch in Google Scholar

Haeng-Soon Lee, PhD

Laboratory of Plant Cell Biotechnology

Korea Research Institute of Bioscience and Biotechnology (KRIBB)

Oun-Dong 52

Yusong-gu

Daejeon 305-806

Korea

Fax: +82-42-860-4608

Email: hslee@kribb.re.kr

References

• 1 Nibbering P H, Ravensbergen E, Welling M M, van Berkel L A, van Berkel P HC, Pauwels E KJ, Nuijens J H. Human lactoferrin and peptides derived from its N terminus are highly effective against infections with antibiotic-resistant bacteria.  Infect Immun. 2001;  69 1469-76
  CrossrefPubMedSearch in Google Scholar
• 2 Soukka T, Tenovuo J, Lenander L M. Fungicidal effect of human lactoferrin against Candida albicans .  FEMS Microbiol Lett. 1992;  69 223-8
  PubMedSearch in Google Scholar
• 3 Zhang G H, Mann D M, Tsai C M. Neutralization of endotoxin in vitro and in vivo by a human lactoferrin-derived peptide.  Infect Immun. 1999;  67 1353-8
  PubMedSearch in Google Scholar
• 4 Hasegawa K, Motsuchi W, Tanaka S, Dosako S. Inhibition with lactoferrin of in vitro infection with human herpes virus.  Jpn J Med Sci Biol. 1994;  47 73-85
  PubMedSearch in Google Scholar
• 5 Ward P P, May G S, Headon D S, Conneely O M. An inducible expression system for the production of human lactoferrin in Aspergillus nidulans .  Gene. 1992;  122 219-23
  CrossrefPubMedSearch in Google Scholar
• 6 Lian Q, Richardson T. Expression and characterization of human lactoferrin in yeast Saccharomyces cerevisiae .  J Agric Food Chem. 1993;  41 1800-7
  CrossrefPubMedSearch in Google Scholar
• 7 van Berkel P HC, Welling M M, Geerts M, van Veen H A, Ravensbergen B, Salaheddine M, Pauwels E KJ, Nuijens J, Nibbering P H. Large scale production of recombinant human lactoferrin in the milk of transgenic cows.  Nature Biotechnol. 2002;  20 484-87
  CrossrefPubMedSearch in Google Scholar
• 8 Arakawa T, Chong D KX, Slattery C W, Langridge W HR. Improvements human health through production of human milk proteins in transgenic food plants.  In: Chemicals via Higher Plant Bioengineering. Shahidi, Editors New York Kluwer academic/Plenum Publishers 1999: 149-59
  Search in Google Scholar
• 9 Mitra A, Zhang Z. Expression of a human lactoferrin cDNA in tobacco cells produces antibacterial protein(s).  Plant Physiol. 1994;  106 977-81
  CrossrefPubMedSearch in Google Scholar
• 10 Salmon V, Legrand D, Slomianny M C, el Yazidi I, Spik G, Gruber V, Bournat P, Olagnier B, Mison D, Theisen M, Meroc B. Production of human lactoferrin in transgenic tobacco plants.  Protein Expr Purif. 1998;  13 127-35
  PubMedSearch in Google Scholar
• 11 Anzai H, Takaiwa F, Katsumata K. Production of human lactoferrin in transgenic plants. In: Shimazaki K, Tsuda H, Tomita M, editors Lactoferrin: Structure, Function and Application. Elsevier science B.V. 2000: 265-71
  Search in Google Scholar
• 12 Chong D K, Langridge W H. Expression of full-length bioactive antimicrobial human lactoferrin in potato plants.  Transgenic Res. 2000;  9 71-8
  CrossrefPubMedSearch in Google Scholar
• 13 Fischer R, Emans N, Schuster F, Hellwig S, Drossard J. Towards molecular farming in the future: using plant-cell-suspension cultures as bioreactors.  Biotechnol Appl Biochem. 1999;  30 109-12
  PubMedSearch in Google Scholar
• 14 Asaka I, Li I, Hirotani M, Asada Y, Furuya T. Production of ginsenosides by culturing ginseng (Panax ginseng) embryogenic tissues in bioreactors.  Biotechnol Lett. 1993;  15 1259-64
  PubMedSearch in Google Scholar
• 15 Proctor J TA, Bailey W G. Ginseng: industry, botany, and culture.  Hortic Rev. 1987;  9 187-236
  PubMedSearch in Google Scholar
• 16 Lee H S, Liu J R, Yang S G, Lee Y H, Lee K W. In vitro flowering of plantlets regenerated from zygotic embryo-derived somatic embryos of ginseng.  HortScience. 1990;  25 1652-4
  PubMedSearch in Google Scholar
• 17 Lee H S, Kim S W, Lee K W, Eriksson T, Liu J R. Agrobacterium-mediated transformation of ginseng (Panax ginseng) and mitotic stability of this the inserted β-glucuronidase gene in regenerants from isolated protoplasts.  Plant Cell Rep. 1995;  14 545-9
  PubMedSearch in Google Scholar
• 18 Murashige T, Skoog F. A revised medium for rapid growth and bioassays with tobacco tissue culture.  Physiol Plant. 1962;  15 473-97
  CrossrefPubMedSearch in Google Scholar
• 19 Kim K Y, Kwon S Y, Lee H S, Hur Y, Bang J W, Kwak S S. A novel oxidative stress-inducible peroxidase promoter from sweet potato: molecular cloning and characterization in transgenic tobacco plants and cultured cells.  Plant Mol Biol. 2003;  51 831-8
  CrossrefPubMedSearch in Google Scholar

Haeng-Soon Lee, PhD

Laboratory of Plant Cell Biotechnology

Korea Research Institute of Bioscience and Biotechnology (KRIBB)

Oun-Dong 52

Yusong-gu

Daejeon 305-806

Korea

Fax: +82-42-860-4608

Email: hslee@kribb.re.kr