JOURNAL OF BIOSCIENCE AND BIOENGINEERING Vol. 100, No. 6, 667–671. 2005 DOI: 10.1263/jbb.100.667
© 2005, The Society for Biotechnology, Japan
Preparation of Silk Protein Sericin as Mitogenic Factor for Better Mammalian Cell Culture Satoshi Terada,1* Masahiro Sasaki,2 Kana Yanagihara,1 and Hideyuki Yamada2 Department of Applied Chemistry and Biotechnology, University of Fukui, 3-9-1 Bunkyo, Fukui 910-8507, Japan1 and Technology Department, Seiren Co. Ltd., 1-10-1 Keya, Fukui 918-8560, Japan2 Received 8 February 2005/Accepted 10 September 2005
We previously reported that sericin small (sericin-S), with a molecular weight that ranges from 5 to 100 kDa, is a cell culture supplement used to accelerate cell proliferation. In this study, a novel preparation method for sericin and several applications of sericin were examined. Sericin large, prepared under nonhydrolyzing conditions and ranging from 50 to 200 kDa, also accelerated cell proliferation, but its effects were inferior to those of sericin-S. Additional sericin preparations with various molecular weights that were differentially hydrolyzed were also tested but none of them was significantly superior to sericin-S, and neither were several recombinant sericin peptides. Sericin-S successfully accelerated the proliferation of hybridoma cells in various serumfree media, implying the mitogenic effect of sericin is independent from media. We also demonstrated that sericin-S successfully induced the proliferation of CTLL-2, an established T lymphocyte cell line, under IL-2 starvation conditions. These results indicate that sericin, particularly sericin-S, improves serum-free mammalian cell culture. [Key words: sericin, silk protein, hybridoma, CTLL-2, proliferation, serum-free medium, IL-2]
(BSA). Hence, the risk of BSE or other infections cannot be avoided when serum-free media supplemented with other proteins that are derived from mammals are used. In addition to fetal bovine serum (FBS), cytokines are another issue. They are very expensive but their bioactivities are often essential for cell culture. As a result, low-price alternatives have attracted increasing interest. We previously reported that hydrolyzed sericin, derived from silkmoth Bombix mori cocoon, is a good supplement for cell culture media in order to accelerate the proliferation of mammalian cells (2, 3). Insect cell culture was also reported to be improved by sericin (4). Sericin was added to freezing media as an alternative to FBS and successfully improved various cell survival during the cryopreservation (5, 6). These findings encouraged us to investigate the use of sericin as an alternative for FBS and cytokines to improve serum-free cultures. In our previous report (2), sericin was prepared in an alkaline solution and this hydrolyzed sericin had a molecular weight of less than 70 kD and was termed sericin-S (sericin small). In the first section of the present study, we attempted to identify a better preparation method for sericin. As a first approach, sericin-S was fractionated using gel-exclusion chromatography. Then, sericin of higher molecular weights was prepared from virgin silk under nonhydrolyzing conditions and was termed sericin-L (sericin large). Several recombinant sericin peptides that were designed from the repetition of sericin amino acid sequences (7) were also tested. In the latter section of this study, improvement of serumfree media and alternatives to cytokines were investigated by using the best preparation of sericin.
Development of mammalian cell culture aims to produce various bioactive proteins such as erythropoietin as a medication for anemia, which cannot be produced by Escherichia coli cells in their active forms. Moreover, ex vivo culture is a pivotal process for cell therapy and for regenerative medicine, especially in the case of skin cells and hemopoietic stem cells, to increase population numbers or to induce differentiation and other phenotypes. Yet current mammalian cell culture systems offer various challenges including the requirement of serum-free culture of particular useful cells. Although serum is an effective factor for some mammalian cell culture types, it can also result in various disadvantages. Serum is frequently contaminated with viruses despite claims that various filters can remove viruses (1), and the risk of bovine spongiform encephalitis (BSE) is also a concern. Serum costs constitute a major part in the cost of cell culture medium: its cost often accounts for more than ten times that of the total cost of the other constituents. To overcome these disadvantages, culturing techniques using serum-free media have been developed. Although various serum-free media are commercially available, most cell lines cultured in serum-free media are inferior both in growth rate and in maximum cell density to those cultured in media supplemented with serum. In addition to the inferiority of the proliferation rate and population numbers, many serum-free media must be supplemented with other proteins derived from mammals such as bovine serum albumin * Corresponding author. e-mail:
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MATERIALS AND METHODS Preparation of sericin Two types of sericin with different molecular weights were used in this study: the hydrolyzed sericinS and sericin-L. Both types were derived from the cocoons of Bombyx mori “Showa”. Sericin-S was prepared following the methods of Kato et al. (8) and its molecular weight ranged from 5 to 100 kDa. The molecular weight of sericin-L ranged from 50 to 200 kDa, and it was prepared by the following procedures. The cocoons of Bombyx mori were heated at 120°C for 30 min and then filtered through a glass microfiber filter (GF/A; Whatman International, Maidstone, UK). The filtrates were dialyzed against deionized water and finally freeze-dried. The amino acid composition of the extracted sericins was analyzed using an amino acid analyzer (LC-10A; Shimadzu, Kyoto). The same preparations of sericin-S and sericin-L were used for all experiments in the present study. Sericin-S was fractionated by gel-exclusion chromatography through a column of Sephacryl S-200HR (Pharmacia Fine Chemicals, Uppsala, Sweden). Molecular weights were estimated using five standards of known molecular weights. Five other sericin preparations varying in molecular weights, and with different levels of hydrolysis, were also prepared under various hydrolysis conditions and purified by gel-exclusion chromatography through a column Sephacryl S-200HR. Recombinant sericin peptides The recombinant sericin peptides SerD and SerT were synthesized in E. coli cells transfected with pQEserD or pQEserT and prepared following the methods of Tsujimoto (7). Briefly, E. coli cells harboring one of these plasmids were sonicated, boiled and centrifuged. The soluble fraction obtained was then purified using Ni-NTA agarose (Qiagen, Valencia, CA, USA) and the elute was treated with factor Xa to obtain mature sericin peptide and this product was purified by HPLC on a C8 column (Amersham Pharmacia Biotech, Buckingham, UK). SerD and SerT are, respectively, a dimer and a tetramer of the peptide with the following amino acid sequence: SSTGS SSNTD SNSNS AGSST SGGSS TYGYS SNSRD GSV. Cell lines and culture conditions The 2E3-O cell line is a mouse hybridoma, which was derived from the mouse myeloma P3X63 AG8U.1 by electric fusion with mouse spleen cells (9). The cells adapted to the serum-free medium ASF103 (Ajinomoto, Tokyo) so well that their growth rate in ASF103 was higher than that in RPMI 1640 medium supplemented with FBS. Because ASF103 contains BSA, BSA-free ASF104 medium (Ajinomoto) was used during the assay of mitogenic activity of sericin. Cells were cultured in either IS MAB-V containing 5 µg⋅ml–1 of recombinant insulin but no components derived from animals (Irvine Scientific, Santa Ana, CA, USA), or in protein-free medium UltraDOMA-PF (Cambrex, East Rutherford, NJ, USA), or in protein-free medium CD Hybridoma (Invitrogen, Carlsbad, CA, USA), or in protein-free medium Hybri-Max (Sigma, St. Louis, MO, USA) and culturing methods were compared. The interleukin 2 (IL-2)-dependent cell line CTLL was also used and cultured in RPMI 1640 medium (Nissui Pharmaceutical, Tokyo) supplemented with 10% FBS (Gibco, Carlsbad, CA, USA), 0.2% sodium bicarbonate (Wako Pure Chemical Industries, Osaka), 10 mM HEPES (Sigma), 2 mM glutamine (Gibco), and 0.06 mg ⋅ml–1 kanamycin. Murine IL-2 was purchased from Cosmo Bio (Tokyo). All cell types were grown in 24-well plates (Sumitomo Bakelite, Tokyo) at 37°C, under humidified air, and 5% CO2. The numbers of viable and dead cells were determined by counting with a hemocytometer under a phase contrast microscope using trypan blue exclusion. Assay of mitogenic activity Although a number of serumfree media are available for mammalian cell culture, most of them
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are inferior to media supplemented with serum. To determine whether sericin is a potent mitogenic supplement to serum-free media, the proliferation of hybridoma cells in various serum-free media was measured and compared to cultures without sericin. Whether sericin can substitute for IL-2 was also tested. The IL-2-dependent cell line CTLL-2 was raised in IL-2 limited conditions supplemented with sericin. After several days in culture, the proliferation of CTLL-2 was measured.
RESULTS AND DISCUSSION Fractionation of sericin-S for a better sericin preparation To find a more effective fraction of sericin, sericin-S was fractionated using gel-chromatography. Three fractions were obtained and their molecular weights were 20–70, 10– 40 and less than 30 kD (Fig. 1a). Differences in molecular weights were confirmed by SDS–polyacrylamide disc gel electrophoresis (data not shown). The fractions were termed fraction L (20–70 kD), fraction M (10–40 kD) and fraction S (less than 30 kD), and their respective mitogenic activities in hybridoma cells were measured and compared (Fig. 1b). In the negative control culture, the number of viable cells was 68,000 cells⋅ ml–1, whereas in the presence of unfractionated sericin-S, proliferation was accelerated and viable cell number was 126,000 cells⋅ ml–1. In the presence of any of the fractionated sericin preparations, proliferation was also accelerated; the numbers of viable cells with fraction L, M and S were 1.28 ⋅105, 99 ⋅105 and 70 ⋅105 cells ⋅ml–1, respectively, indicating that sericin with a greater molecular
FIG. 1. Fractionation of sericin and mitogenic activity of resulting fractions. Sericin was fractionated by chromatography through a Sephacryl S-200HR column (Pharmacia). (a) Gel-chromatography of sericin fractions. Peaks: 1, sericin-S fraction L; 2, fraction M; 3, fraction S. (b) Hybridoma cells (20,600 cells) in 1 ml of ASF104 medium were seeded into each well of a 24-well plate and were cultured for 39 h, and viable cell density was determined. 1, Control; 2, 0.1% sericin-S (
