Silk Protein Sericin : as a Novel Mitogenic Supplement for

Mammalian Cell Culture

On-Line Number 618
Kana Yanagihara,1 Satoshi Terada,1 Tomohiro Toyosawa,1 Taeko Nishimura,1 Tomoaki Kumagai,1
Masao Miki,1 Masahiro Sasaki,2 Hideyuki Yamada2
1
Department of Applied Chemistry and Biotechnology, Faculty of Engineering, University of Fukui,
3-9-1, Bunkyo, Fukui, 910-8507, Japan E-mail: terada@acbio.fukui-u.ac.jp
2
Research Department, Seiren Co. Ltd., 1-10-1, Keya, Fukui 918-8560, Japan E-mail: m.sasaki@seiren.
co.jp

ABSTRACT
Although many bio-active proteins and vectors for gene therapy are produced by mammalian cell culture,
its efficiency should be improved. Culture media are very expensive because serum or its derivatives are
essential for them, and so there is concern about the risk such as BSE. Therefore inexpensive, safe and
potent mitogenic supplement for mammalian cell culture is eagerly desired.
In this study, sericin, a protein derived from silkworm, was considered as such a factor. In the presence of
sericin, the proliferation of all mammalian cell lines tested were accelerated. In comparison with bovine
serum albumin, a widely used supplement in serum-free medium, sericin had better effect on the
proliferation of human hepatoma HepG2. Although most of proteins are easily inactivated by autoclave
sterilization, mitogenic activity of sericin was not damaged after autoclave. As well as sericin derived
from silkworm, recombinant sericin peptide synthesized by E.coli stimulated the proliferation of
mammalian cells.
In cell therapy or bio-artificial organs, stable storage and supply of the cells are crucial. Therefore the
effect of sericin on cryopreservation was tested. Sericin was added to the freezing medium and HepG2
cells, which several groups have used in bio-artificial liver, were frozen at -80 C for three days. Cells
were thawed out, cultured for one day and the viable cell number was determined. Supplementation of
sericin improved the viable cell number by 27%.
These results indicate that sericin is a novel and suitable mitogenic supplement for mammalian cell
culture as well as supplement for cyropreservation.

KEYWORDS
sericin, proliferation, cyropreservation, silk

INTRODUCTION
Mammalian cells are industrially cultured in order to produce bio-active proteins or vectors for gene
therapy or to be used for cell therapy, etc. Mammalian cells usually require serum or its replacement in
the culture medium. The sera used most frequently is a fetal bovine serum (FBS), which is frequently
contaminated with viruses, and even the risk of prions, or bovine spongiform encephalopathy (BSE),
cannot be erased. Serum also contains identified and unknown factors outside the operator's control,
indicating that the presence of serum is the principal obstacle to purification for the recovery of cell
products and limits pharmaceutical acceptance of the product. Therefore inexpensive, safe and potent
mitogenic supplement for mammalian cell culture is eagerly desired.
In this study, sericin was considered as such a factor. Sericin, together with fibroin, is one of the two
major components of raw silk. While fibroin is the predominant component, sericin constitutes about
quarter and minor components are salt and wax that might act as a water repellent for the cocoon. Sericin

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is a gummy coating on raw-silk filaments and it is removed by a treatment called degumming in order to
make the silk lustrous and semitransparent (Fig.1). The degumming treatment is essentially an alkaline
scouring operation and is carried out at a temperature of about 95C. In this process, sericin is degraded
and solubilized in water and has been abolished.
Recently, several functions of sericin have been revealed and novel applications have been proposed.
Sericin acts as antioxidant to inhibit tyrosinase and lipid peroxidation (Kato, et al., 1998), suggesting the
utility in cosmetics. Since oxidative stress could induce carcinogenesis, dietary sericin could inhibit
carcinogenesis. For example, feeding diets containing sericin to mice suppressed colon carcinogenesis
induced by 1,2-dimethylhydrazine (Sasaki, et al., 2000a). Enhancement of the bioavailability of several
metal ions in rats was also suggested during consumption of sericin (Sasaki, et al., 2000b). A recombinant
sericin peptide protected a bacteria against freezing stress (Tsujimoto, et al., 2001).

Filature
others
Raw silk Ser
Ala
Weave
Degumming
Thr

Gly Asp
Silk
Sericin

Figure 1. Sericin was waste material from

degumming process. Figure 2. Molar ratio of amino acid in the
obtained sericin hydrolysate

MATERIALS AND METHODS

Preparation of sericin
Sericin from the cocoons of silkworm was prepared by the following procedures. Ten liters of 0.2%
sodium carbonate solution (pH 11 - 12) containing 200 g of the cocoon was heated at 95 C for 120
minutes and then filtered through a glass microfiber filter to remove the fibroin. The filtrate was dialyzed
against deionized water and dried by spray drying. The amino acid composition of the extracted sericin
was analyzed using an amino acid analyzer (LC-10A, Shimazu, Japan).
The recombinant sericin peptides were synthesized in E. coli transfected with pQEserT or pQEserD and

2
prepared by the method of Tsujimoto (Tsujimoto, et al., 2001).

Cell Lines and Culture Conditions

The hybridoma cell line 2E3-O was cultured in serum-free ASF103, serum- and BSA-free ASF104
(Ajinomoto, Japan) or RPMI 1640 medium (Nissui, Japan) supplemented with 10% FBS (vol/vol), 20
mM HEPES, 0.2% NaHCO3, 2 mM glutamine and 0.06 mg ml-1 kanamycin. HepG2 was cultured in
serum- and BSA-free ASF104 or DMEM medium (Nissui, Japan) supplemented with 10% FBS (vol/vol),
20 mM HEPES, 0.2% NaHCO3, 2 mM glutamine and 0.06 mg ml-1 kanamycin. The cells were grown in
24 well plates (Asahi Techno Glass Co., Japan) at 37 in humidified air containing 5% CO2.
The numbers of viable and dead cells were determined by counting in a hemocytometer under a phase
contrast microscope using trypan blue exclusion.

Cryo-preservation

HepG2 culture growing exponentially was collected and centrifuged. The growth medium was exchanged
into freezing medium. Freezing medium was DMEM containing 10% DMSO supplemented with 10%
FBS, or 0.1% sericin, or without any supplement. After being frozen for three days at -80C, cells were
thawed and seeded into growth medium (DMEM supplemented with 10% FBS). On the next day, viable
cell numbers were determined.

RESULTS AND DISCUSSION

Amino acid composition of sericin

For the validation of sericin hydrolysate obtained from degumming process, the amino acid composition
was analyzed using an amino acid analyzer. As shown in Figure 2, molar percentage of serin was 32%,
indicating that contamination of other proteins such as fibroin was little and that most of obtained
polypeptides are derived from sericin.

Sericin accelerated the proliferation of a hybridoma cell line

In order to investigate the effect on proliferation, sericin was supplemented to the culture of the
hybridomas for three days and then the cell density was determined. As shown in Figure 3, culture in the
presence of 0.1% sericin significantly increased the cell number. The culture medium was serum-free
media ASF104 (Ajinomoto, Japan), where the hybridoma grow better than in media supplemented with
FBS. Sericin also accerelated the proliferation rates of various mammalian cell lines (Terada, et al., 2002)
and an insect cell line Sf-9 (Takahashi, 2003).
Most proteins are easily denatured and inactivated by heating, but the sericin was purified by a procedure
including treatment with boiling water (95C), suggesting that additional application of heat would not
inactivate sericin. Due to this idea, sericin exposed to the temperature required for autoclaving (121C)
was also tested. Autoclaved sericin also increased the hybridoma population equally, irrespective of the
sterilization method. This result indicates that autoclaving did not affect the activity of sericin and that
sericin would be useful because autoclaving is free from Mycoplasma contamination.

Comparison of sericin with bovine serum albumin as mitogenic supplement

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In order to characterize sericin as mitogen, the effect of sericin was compared with bovine serum albumin
(BSA), that has been widely supplemented into serum-free media. Human hepatocarcinoma HepG2 is
usually cultured in DMEM supplemented with 10% FBS.
268,000 HepG2 cells were seeded into each well and cultured for one day for attachment on the wells.
Then medium was changed into ASF 104 serum-free medium supplemented with sericin or BSA or
without and they were cultured for four days. As shown in Figure 4, sericin significantly accerelated the
proliferation, while BSA failed to improve the culture. This result shows that sericin would be a better
mitogenic supplement than BSA. Although sericin was effecctive mitogen, mitogenic activity of FBS was
superior to that of sericin. The effect of higher concentration of sericin such as 1% was inferior to that of
0.1% sericin. Although the mechanism of sericin is unkwon, the mitogenic activity depends on the
molecular weight range of the sericin hydrolysate (unpublished data), suggesting that some of
polypeptides activate the receptors on cell surfaces.

500000
Viable cell density (cells/ml)

400000

300000

200000

100000

0
blank 0.1% Sericin, 0.1% Sericin,
filtrated autoclaved

Figure 3. Effect of sericin on the proliferation of a hybridoma cell line 2E3-O in ASF104 (N=3)

Recombinant sericin peptides

Tsujimoto et al. designed the sericin peptide from the sericin-rich internally repetitive 38-mer sequence of
sericin and constructed expression plasmids pQEserD and pQEserT (Tsujimoto, et al., 2003). pQEserD
and pQEserT encode dimer and tetramer of the consensus sequence of sericin, respectively. The dimer
peptide SerD and the tetramer SerT were successfully expressed in E. coli and purified.
Hybridoma cells were seeded in ASF104 medium with 0.03% sericin peptide and cultured for three days.
As shown in table 1, both dimer and tetramer sericin peptides significantly accelerated the proliferation.

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 3000000

Viable cell number

2000000

1000000

0
N. C. Sericin BSA
0.1% 0.1%

Figure 4. Effect of sericin on the proliferation of a hepatoma cell line HepG2 in ASF104 (N=3)

Position 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38

S S T D A S S N T D S N S N S A G S S T S G G R R T Y G Y S S N S R D G S V
S S T G S S S N T D S N S S N A G S S T S G G S S T Y G Y S S N S R D G S V
S T T G S S S N T D S N S N S V G S R R S G G S S S H E D S S K S R D E N V
S T T G S S S N T D S N S N S V G S S T S G G R R T Y G Y S S N S R D G S V
S S T G S S S N T D S N S N S V G S S T S G G S S T Y G Y S S N S R D G S V
S S T G S S S N T D S N S N S A G S S T S G G S S T Y G Y S S N S H D G S V

Consensus S S T G S S S N T D S N S N S A G S S T S G G S S T Y G Y S S N S R D G S V
(sericin peptide)

Figure 5. Design of 38-mer sequence of the recombinant sericin peptide

Table 1. Effect of recombinant sericin peptide on the proliferation of hybridoma

Nagative control Sericin peptide, dimer Sericin peptide, tetramer
Cell number
4.88 0.50 6.69 0.50 6.06 0.68
increase

Effect of sericin during cryo-preservation

Tsujimoto et al. reported that sericin had cryoprotective effect on E. coli. Sericin was added into freezing
medium and after thawed, the vaiable cell number was compared with FBS addition. After three days of
cryopreservation at -80C, the viable cell number was reduced in the absence of sericin (Figure 6).
Cryopreservation with sericin increased the viable cell number, and sericin had a more remarkable effect
than FBS. After thaw, sericin was removed and the cells were cultured without sericin, suggesting that
sericin reduced damage during freezing. This result suggests that sericin would be a preferable factor for
cryopreservation.

5
 1500000
Viable cell number
1000000

500000

0
N. C. 10% FBS 0.1% sericin

Figure 6. Effect of sericin on viable cell number after cryopreservation

CONCLUSIONS
Inexpensive, safe and potent mitogenic supplement for mammalian cell culture is eagerly desired. Here,
sericin derived from silkworm coccoon was tested. In the presence of sericin, the proliferation of various
mammalian cell lines were accelerated As well as filtrated sericin, autoclaved sericin had equivalent
effect on the proliferation. Addition of sericin into freezing medium improved the viable cell number after
thawing. These results indicate that sericin is useful as a mitogenic supplement for mammalian cell
culture and as a supplement for cyropreservation.

REFERENCES
Kato, N., S. Sato, A. Yamanaka, H. Yamada, N. Fuwa and M. Nomura; " Silk protein, sericin, inhibits
lipid peroxidation and tyrosinase activity. " Biosci. Biotechnol. Biochem. 62. 145-147 (1998)
Sasaki, M., N. Kato, H. Watanabe and H. Yamada; " Silk protein, sericin, suppresses colon carcinogenesis
induced by 1,2-dimethylhydrazine in mice. " Oncol. Rep. 7, 1049-1052 (2000a)
Sasaki, M., H. Yamada and N. Kato " Consumption of silk protein, sericin elevates intestinal absorption
of zinc, iron, magnesium and calcium in rats. " Nutr. Res. 20, 1505-1511 (2000b)
Takahashi, M., K. Tsujimoto, H. Yamada, H. Takagi and S. Nakamori ; " The silk protein, sericin, protects
against cell death caused by acute serum deprivation in insect cell culture " Biotech. Lett. 25.
1805-1809 (2003)
Terada, S., T. Nishimura, M. Sasaki, H. Yamada, M. Miki ; " Sercin, a Protein Derived from Silkworms,
Accelerates the Proliferation of Several Mammalian Cell Lines Including a Hybridoma " Cytotech. 40,
3-12 (2002)
Tsujimoto, K., H. Takagi, M. Takahashi, H. Yamada and S. Nakamori; " Cryoprotective effect of the
serine-rich repetitive sequence in silk protein sericin." J. Biochem. 129, 979-986 (2001)