Optimal silkworm larva host for high-level production of Mus musculus IL-4 using a baculovirus expression vector system

Journal Pre-proofs Full length article Optimal silkworm larva host for high-level production of Mus musculus IL-4 using a baculovirus expression vector system Masahiko Kobayashi, Jian Xu, Kohei Kakino, Akitsu Masuda, Masato Hino, Naoki Fujimoto, Kosuke Minamihata, Noriho Kamiya, Hiroaki Mon, Hiroshi Iida, Masateru Takahashi, Takahiro Kusakabe, Jae Man Lee PII: DOI: Reference:

S1226-8615(19)30727-7 https://doi.org/10.1016/j.aspen.2019.12.014 ASPEN 1491

To appear in:

Journal of Asia-Pacific Entomology

Received Date: Revised Date: Accepted Date:

7 November 2019 21 December 2019 26 December 2019

Please cite this article as: M. Kobayashi, J. Xu, K. Kakino, A. Masuda, M. Hino, N. Fujimoto, K. Minamihata, N. Kamiya, H. Mon, H. Iida, M. Takahashi, T. Kusakabe, J. Man Lee, Optimal silkworm larva host for high-level production of Mus musculus IL-4 using a baculovirus expression vector system, Journal of Asia-Pacific Entomology (2019), doi: https://doi.org/10.1016/j.aspen.2019.12.014

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Optimal silkworm larva host for high-level production of Mus musculus IL-4 using a baculovirus expression vector system

Masahiko Kobayashia, Jian Xua, Kohei Kakinoa, Akitsu Masudaa, Masato Hinoa, Naoki Fujimotob, Kosuke Minamihatab, Noriho Kamiyab,c, Hiroaki Mona, Hiroshi Iidad, Masateru Takahashie, Takahiro Kusakabea, Jae Man Leef,*,[email protected] aLaboratory

of Insect Genome Science, Kyushu University Graduate School of

Bioresource and Bioenvironmental Sciences, Motooka 744, Nishi-ku, Fukuoka 819-0395, Japan bDepartment

of Applied Chemistry, Graduate School of Engineering, Kyushu

University, Motooka 744, Nishi-ku, Fukuoka 819-0395, Japan cDivision

of Biotechnology, Center for Future Chemistry, Kyushu University, Motooka

744, Nishi-ku, Fukuoka 819-0395, Japan dLaboratory

of Zoology, Graduate School of Agriculture, Kyushu University, Motooka

744, Nishi-ku, Fukuoka 819-0395, Japan eLaboratory

of DNA Replication and Recombination, Division of Biological and

Environmental Sciences and Engineering, King Abdullah University of Science and Technology, 4700 KAUST Thuwal, Jeddah 23955, Saudi Arabia fLaboratory

of Creative Science for Insect Industries, Kyushu University Graduate

School of Bioresource and Bioenvironmental Sciences, Motooka 744, Nishi-ku, Fukuoka 819-0395, Japan *Corresponding

author.

Graphical abstract Highlights Recombinant MmIL-4 was expressed and purified by the silkworm-BEVS

1

C-tagged rMmIL-4 is a better construct to produce rMmIL-4 in the silkworm-BEVS. We found that the n70 strain produced the highest amount of rMmIL-4 among 19 silkworm strains.

Abstract Interleukine-4 (IL-4) is a cytokine that plays an important role in the immune system and recognized as a biological medicine. Therefore, there is a demand for the production of IL-4 with high performance. The expression of a recombinant IL-4 protein in the prokaryotic system usually results in the formation of an inclusion body. To date, the solution to obtain those active products without the refolding process remains to be established. In this study, we tried to acquire a biologically active recombinant Mus musculus IL-4 (rMmIL-4) using a silkworm-baculovirus expression vector system (silkworm-BEVS). We constructed two recombinant baculoviruses coding rMmIL-4 with the distinct location of affinity purification tags and succeeded in the expression and purification of rMmIL-4 proteins directly without the refolding process. Both purified proteins displayed comparable biological activity to the commercial proteins produced by the E. coli expression system. Besides, we performed screening of silkworm strains to seek optimal hosts for the mass-production of rMmIL-4. Intriguingly, we found that some silkworm strains showed significantly higher secretion levels of rMmIL-4 in silkworm sera. Our study provides meaningful insights into the industrial-scale production of rMmIL-4 with high productivity for pharmaceutical 2

applications in the future. Keywords: Silkworm expression system; MmIL-4; Purification; Baculovirus

1. Introduction Interleukin-4 (IL-4), also called B-cell stimulatory factor-1 (BSF-1), is a cytokine mainly produced by T helper 2 (Th2) cells, mast cells, and basophils. It regulates the differentiation from naïve T cells to Th2 cells and facilitates humoral immune responses (Sokol et al., 2008; Elo et al., 2010; Paul, 2015). IL-4 is also known as a multifunctional cytokine that plays critical roles in the regulation of immune responses against a variety of extracellular stimuli (e.g., parasites and allergens) (Nelms et al., 1999; Steinke and Borish, 2001; Finkelman et al., 2004). As a commercialized medicine worldwide, IL-4 treatment has been proven to be effective against psoriasis, one of the autoimmune diseases (Ghoreschi et al., 2003). Besides, it has been also reported that various cytokines, including IL-4, can be utilized as promising adjuvants to enhance immune responses (Calarota et al., 2008; Xu et al., 2008; Castaldello et al., 2010). As an example, recently, Feng et al. reported that a fusokine by fusing IL-4 and granulocyte-macrophage colony-stimulating factor (GM-CSF) to be decorated on the surface of virus-like particles enhances immunogenicity significantly (Feng et al., 2015). Based on this context, it is of great interest to efficiently obtain a large amount of IL-4 for either research or clinical use in the market. To date, recombinant Mus musculus IL-4 (rMmIL-4) has been expressed in several 3

expression systems using various hosts. For instance, in Escherichia coli, up to 100 mg of rMmIL-4 is produced from 1 L fermentation (Levine et al., 1995). In transgenic rice seeds, 2.2 mg of rMmIL-4 is obtained from 80 g rice grain powder (Fujiwara et al., 2016). By looking into the rMmIL-4 proteins expressed in the above systems, however, it mainly results in the formation of an inclusion body and requires the refolding process, which makes it an extremely challenging issue to produce rMmIL-4 in a large-scale manner. Previously, it has been reported that cultured cell-based baculovirus expression vector system (BEVS) could produce functional murine IL-4 proteins without refolding process in culture media (Rodewald et al., 1990; Cottrez et al., 1994). This method, however, was reported to be suitable for experiments but not industrial purposes. Thus, in this study, we tried to produce high-level rMmIL-4 using silkworm-BEVS. Silkworm-BEVS is widely used to produce eukaryotic recombinant proteins, because, in most cases, silkworm can produce a considerable amount of recombinant proteins compared to the cultured insect cells or mammalian cells (Kato et al., 2010; Morifuji et al., 2018). Additionally, this system is sometimes able to avoid the formation of the inclusion body (Kinoshita et al., 2019). Thus, our group, previously, has established an efficient production procedure by the silkworm-BEVS and successfully produced a variety of proteins that are difficult to express in E. coli (Hino et al., 2016; Masuda et al., 2018). In this study, we successfully expressed rMmIL-4 by the silkworm-BEVS and established a simple two-step affinity purification procedure without the protein refolding processes. Furthermore, the biological activity assay revealed that the 4

bioactivity of rMmIL-4 is comparable to the commercial rMmIL-4 derived from E. coli. Besides, to enhance the productivity of rMmIL-4 in a silkworm larva, we screened 19 silkworm strains with distinct genetic backgrounds to compare the expression levels and discovered several high productive strains to express rMmIL-4 proteins. To sum up, we established how to easily obtain biologically active rMmIL-4 by using the silkworm-BEVS and the results presented here indicated that this system should be a powerful method for the efficient production of rMmIL-4.

2. Materials and Methods

2.1. Cells and Silkworms The NIAS-Bm-oyanagi2 cells, BmO2, were kindly provided by Dr. Imanishi and cultured in IPL-41 medium (Sigma, St. Louis, MO) with 10% fetal bovine serum (Gibco, Grand Island, NY) at 27 °C. d17 and other 18 strains of silkworm larvae used in this study were provided by the Institute of Genetic Resources, Kyushu University Graduate School, supported by the Japan National BioResource Project. Detailed information

for

these

silkworm

strains

could

be

found

at

https://shigen.nig.ac.jp/silkwormbase/about_kaiko.jsp. The silkworm larvae were reared on fresh mulberry leaves under controlled environmental conditions at 25–27 °C.

2.2. Construction of Recombinant Baculoviruses Total RNAs from the kidney of ICR mice (Charles River, Japan) were used for 5

first-strand cDNA synthesis with SuperScript II RNase H-reverse transcriptase (Invitrogen, Carlsbad, CA) and oligo-(dT) primer. To construct gateway-based entry clones, open-reading frame (ORF), excluding the native signal peptide (NSP) region (amino acids 1–20), of Mus musculus interleukin 4 (MmIL-4, amino acids 21–140: GenBank accession number, NM_021283) was amplified by polymerase chain reaction (PCR) with KOD-Plus-Neo DNA polymerase (TOYOBO, Tokyo, Japan). The primers used

for

the

PCR

reactions

(5′-CATATCCACGGATGCGACAAAAATCACTTG-3′)

were and

MmIL-4-5-NOSP MmIL-4-3-XhoI

(5′-CCCCCTCGAGGACGAGTAATCCATTTGCATG-3′). The amplicon was digested with XhoI and inserted into the modified pENTR11 (pENTRL2130KdH8STREPTEV or pENTRL2130KTEVdH8STREP, our laboratory stocks) vectors with the EcoRV and XhoI double-digested by Ligation High (TOYOBO, Tokyo, Japan) (Fig. 1A). Both of the modified pENTR11 vectors contain a lobster L21 sequence at 5′ upstream of ORF to enhance

translations

and

the

silkworm

30K

signal

peptide

(MRLTLFAFVLAVCALASNA) to secrete the rMmIL-4 proteins in the media or silkworm sera. Additionally, we added double histidine-tags (H8-tag and H6-tag), an eight amino acid Strep-tag, and a tobacco etch virus (TEV) protease cleavage site at either N-terminus or C-terminus of the ORF of MmIL-4. The resulting constructs were named

pENTR-L2130K-dH8STREPTEV-rMmIL-4

and

pENTR-L2130K-rMmIL-4-TEVdH8STREP, respectively. Subsequently, two rMmIL4 entry vectors and pDEST8 vector (Invitrogen, Carlsbad, CA) were used for the Gateway LR reaction to generate recombinant baculovirus transfer plasmids, according to the 6

manufacturer’s protocol. Recombinant baculovirus was generated using BmNPV/T3 bacmid DNA as described previously (Ono et al., 2007). Each of the generated bacmid DNA was transfected into the BmO2 cells using FuGENE HD transfection reagent (Promega,

Madison,

WI)

to

produce

BmNPV/polh-30K-dH8STREPTEV-rMmIL-4

the

recombinant (N-tagged

virus rMmIL-4)

particles, and

BmNPV/polh-30K-rMmIL-4-TEVdH8STREP (C-tagged rMmIL-4), respectively. On the 4th day after transfection, the culture medium containing recombinant P1 viruses was harvested and centrifuged at 1,000 rpm for 10 min. The supernatant was then collected and kept at 4 °C in the dark. High-titer virus (P3) stock was prepared by a serial infection following the protocols described in the manufacturer’s manual (Invitrogen, USA).

2.3. SDS-PAGE and Western blotting The expressed rMmIL-4 proteins were electrophoresed on 15% sodium dodecyl sulfated-polyacrylamide gel electrophoresis (SDS-PAGE) under the reducing condition and visualized by Coomassie Brilliant Blue (CBB) R-250 staining or used for Western blotting. For Western blotting, after electrophoresis, the samples were transferred onto a polyvinylidene difluoride (PVDF) membrane (Millipore, Milford, MA) and blocked in the TBS-T buffer (20 mM Tris–HCl pH 7.6, 500 mM NaCl, 0.1% w/v Tween-20) with 5% w/v skim milk (Wako, Japan). The membrane was subsequently incubated with HisProbe-HRP (Thermo Scientific, USA) diluted (1:5000) in TBS-T buffer. The membrane was then washed, and the HRP signals were visualized using Super Signal 7

West Pico Chemiluminescent Substrate (Thermo Scientific, USA).

2.4. Viral Infection and Expression Verification of rMmIL-4 in Cells and Silkworm Larvae 6

To verify the expression (time-course experiments) in BmO2 cells, 2 × 10 cells per six-well were infected with recombinant viruses at a multiplicity of infection (MOI) of 1. The infected cells were then harvested at indicated days (3–5) post-infection (dpi), and the culture media were collected, followed by centrifugation at 1000 rpm for 10 min at 4 °C. To verify the expression in the silkworm-BEVS, the recombinant viruses (1 × 105 plaque-forming unit per larvae) were injected into the 5th instar silkworm larvae (day 3) of the d17 silkworm strain. On the indicated days (3–5) after inoculation, the sera were collected into a tube containing 20 mM 1-phenyl-2-thiourea by cutting larval legs and, then, centrifuged at 8,500 rpm for 30 min at 4 °C. The culture media and the supernatant of sera were subjected to Western blotting analysis to detect the expressions of rMmIL-4.

2.5. Purification of rMmIL-4 proteins For the purification of rMmIL-4, a two-step purification procedure was performed by using both His- and Strep-tag chromatography. Briefly, the supernatant of serum was diluted by binding buffer (20 mM Tris–HCl pH 7.4, 0.5 M NaCl, and 20 mM 1-phenyl-2-thiourea), centrifuged at 22,000 rpm for 30 min at 4 °C, and filtered using a 0.45 μm filter (Millipore, USA). The cleared solutions were loaded onto a 5 mL 8

HisTrap excel column (GE Healthcare Bioscience, Piscataway, NJ). After washing with 25 mL of the washing buffer (20 mM Tris-HCl pH7.5, and 0.5M NaCl) containing 30 mM imidazole, the proteins were eluted with 12 mL and 15 mL elution buffer (20 mM Tris-HCl pH7.5, and 0.5M NaCl) containing 100 mM and 500 mM imidazole, respectively. Subsequently, fractions containing rMmIL-4 were concentrated to 8 mL by ultrafiltration using Amicon Ultra-15 3 K filters (Millipore, USA). And the concentrated rMmIL-4 proteins were then diluted up to 50 mL with PBS and loaded onto a 5 mL StrepTrap HP columns (IBA GmbH, Germany). The column was first washed with 25 mL of PBS, and the proteins were eluted with 21 mL of PBS supplemented with 2.5 mM desthiobiotin. Elution fractions were concentrated to 2 mL by ultrafiltration using Amicon Ultra-15 3 K filters (Millipore, USA). The final protein concentration

of

rMmIL-4

was

quantified

by

YabGelImage

software

(https://sites.google.com/site/yabgel/) using bovine serum albumin (BSA) as a standard.

2.6. Bioassay of rMmIL-4 activity The biological activity of the purified rMmIL-4 proteins was measured by using Murine Helper T-cell-derived HT-2 cells (clone A5E; ATCC CRL-1841), which can be used for the proliferation assay of MmIL-2/MmIL-4 (Fernandez-botran et al. 1986). The cells were maintained in RPMI1640 medium (Thermo Fisher Scientific), supplemented with 10% fetal bovine serum (FBS, Thermo Fisher Scientific), 1 % Antibiotic-Antimycotic (Thermo Fisher Scientific), 0.05 mM 2-mercaptoethanol, and the commercial rMmIL-2 (Peprotech, 2 ng/mL) at 37 °C with 5% CO2. The cells were washed with ice-cold 9

1×PBS for 3 times to remove rMmIL-2 in the medium. The cells were then resuspended in the RPMI1640 medium without rMmIL-2 and seeded in a 96-well plate at a density 4

of 1 × 10 cells per well. Subsequently, 100 μL/well of the serially 10-fold-dilutied of silkworm-derived or E. coli-derived commercial rMmIL-4 (R&D Systems) were added to each well. After incubation for 72 h, cell viability was measured by using Cell Counting Kit-8 assay (Dojindo, Japan) according to the protocol provided by the manufacturer. The data were represented by the means ± standard error of five values obtained independently.

2.7. Screening of high productive silkworm strains to produce rMmIL-4 To further discover the suitable strains for the mass-production of rMmIL-4, 19 silkworm strains were employed for the small-scale screening analyses. The C-tagged rMmIL-4 baculovirus (1 × 105 pfu per larva) was used to infect each fifth-instar silkworm larva (day 3), and the sera of each silkworm were collected on ice separately on the 4 dpi. After centrifugation at 8,500 rpm for 30 min, the supernatants were analyzed by Western blotting.

3. Results and Discussions

3.1. Construction of BmNPVs for the production of rMmIL-4 Murine IL-4 gene encodes 141 amino acid residues and includes a native signal peptide (NSP) (aa 1–20) at N-terminus, which is known as a secretion signal. To enhance the 10

secretory efficiency in the silkworm-BEVS, we removed the NSP from MmIL-4 ORF and fused the rest to a silkworm native secretion signal, 30K SP (Fig. 1A). The 30K SP is derived from silkworm 30 kDa protein, which was reported to exhibit a high secretion level in the silkworm-BEVS (Soejima et al., 2013). Additionally, to easily detect and purify the recombinant proteins, Polyhistidine- and Strep-tag were fused to either N-terminus or C-terminus of rMmIL-4. Two distinct constructs are designed to investigate the effects of the tag position on their biological activity. Besides, both constructs used in this study contain a lobster L21 sequence at 5´ UTR, which is known as a translational enhancer. The resulting expression cassettes were transferred to recombinant Bacmid DNA and transfected those bacmids into cultured silkworm cells to generate recombinant BmNPVs.

3.2. Expression of rMmIL-4 in Silkworm Cultured Cells and Silkworm Larvae To investigate an optimal condition to produce rMmIL-4, we performed the time-course analysis of secreted rMmIL-4 proteins expressed by each construct both in the cultured silkworm cells, BmO2, and silkworm larvae, d17 strain. At the time points of 3, 4, and 5 days post-infection (dpi), we harvested the cell media and serum and evaluated the secretion levels of rMmIL-4 on SDS-PAGE. CBB stained gels show whole proteins in the cell media or serum (Fig. 1B, upper panel), and rMmIL-4 proteins were detected by Western Blotting (Fig. 1B, down panel). The expressions of rMmIL-4 increased along with the infection stages of baculovirus, and we found that 4 dpi and 5 dpi are the highest in both BmO2 cells and d17 larvae. At 5 dpi, however, we noticed that some of 11

virus-infected silkworms started dying, probably because of the serious baculovirus virulence. Therefore, to avoid protein degradation due to silkworm death, we decided to collect the sera at 4 dpi to maximize the final yield of rMmIL-4. Interestingly, we found that C-tagged rMmIL-4 are more secreted in the media and sera than N-tagged rMmIL-4, suggesting that C-tagged rMmIL-4 is a better tagging strategy for the mass-production of rMmIL-4 in the silkworm-BEVS.

3.3. Purification of rMmIL-4 To obtain an ideal amount of rMmIL-4, we increased the scale of expression and collected 10 mL sera from about 30 baculovirus-infected d17 silkworm larvae. As indicated in Figure 2A and B, rMmIL-4 protein bands are observed in the crude serum sample (Lane IP), implying that rMmIL-4s were accurately expressed and secreted into the serum. To exclude other impurity proteins efficiently, silkworm sera are subjected to two-step purification using Polyhistidine- and Strep-tag affinity chromatography as described in “Materials and Methods” (Fig. 2). This two-step purification scheme has been reported to be a relatively fast and efficient procedure for the recombinant protein purification (Cass et al., 2005). After concentration, as quantified using bovine serum albumin (BSA) as standard, the yield of purified N- and C- tagged rMmIL-4 are 0.6 mg and 1.1 mg from 10 mL serum, respectively (as described in Supplementary Fig. 1). Taking the productivity into the consideration, we concluded that C-tagged rMmIL-4 is a better construct to produce rMmIL-4 in the silkworm-BEVS.

12

3.4. Biological activity Murine helper T cell that derived HT-2 cell line has been utilized to determine the biological activity of murine IL-2/IL-4, because this proliferation depends on the concentration of MmIL-2/MmIL-4 in medium (Fernandez-Botran et al., 1986; Ramanathan et al., 1989). For comparison, we used the commercial rMmIL-4 derived from E. coli. As shown in Fig. 3, silkworm-derived rMmIL-4s shows higher biological activity than commercial MmIL-4 does when a high (>10 ng/mL) concentration of the IL-4 protein was applied. On the other hand, when at low (<10 ng/mL) concentration, the activity of commercial MmIL-4 is superior to that of rMmIL-4 performs. In the sense of that the general range of working concentration of IL-4 protein, for instant, human IL-4 is around 10-50 ng/mL, the activity of purified rMmIL-4 proteins from this study is comparable to that of commercial MmIL-4. It is known that rMmIL-4 expressed in E. coli needs to go through the refolding process (Levine et al., 1995). In contrast, rMmIL-4 produced by the silkworm-BEVS does not need the refolding process because it is expressed as a soluble form. Although cultured cell-based BEVS also could yield ~1 mg/L functional murine IL-4 in the cultured supernatant, it is not suitable for stable large-scale productions in the industry (Rodewald, et al., 1990). These results revealed that the silkworm-BEVS would be a more powerful platform to produce rMmIL-4 in a large-scale. Furthermore, these results also provided other evidence that the location of fusion-tags might affect the expression level of proteins of interest (POIs), although the activity of both products seems similar. As a result, taking the productivity into consideration, C-tagged rMmIL-4 is an ideal construction for the 13

production of rMmIL-4 in silkworm-BEVS. Therefore, C-tagged rMmIL-4 was used in the later experiments.

3.5. Screening of optimal strains for mass-production of rMmIL-4 Our silkworm stock conserves more than 400 silkworm strains that are genetically distinct, and they are available for screening proper hosts to express the recombinant proteins by the silkworm-BEVS. In our previous study, it is revealed that the expression and secretion levels of recombinant proteins vary by a host silkworm strain (Kawakami et al., 2008; Mitsudome et al., 2014; Masuda et al., 2015). Thus, to improve the productivity, we performed a small-scale screening by using 19 silkworm strains infected with the recombinant baculoviruses encoding C-tagged rMmIL-4. Figure 4A shows the expressions of rMmIL-4 in the different silkworm strains. Compared to the d17 strain, which we first used for the purification, we found that some strains showed higher secretion levels of rMmIL-4 than the d17 strain did. Especially, e10, n22, and n70 strains produced an apparent higher amount of rMmIL-4 proteins. To further determine and compare the final yield, we purified rMmIL-4 using the above-described procedures performed for the d17. Each purified rMmIL-4 was concentrated to a final volume of 5 mL and subjected to SDS-PAGE, and the final yields were evaluated (Fig. 4B and C). We found that the n70 strain produced the highest amount of rMmIL-4 among 19 silkworm strains. The d17 strain was previously reported as one of the most hypersensitive strains to a baculoviral infection, and it is considered to be suitable for the mass-production of POIs in the silkworm-BEVS. However, in this study we 14

discovered that n70 is the best strain to produce rMmIL-4. Only by changing the employed silkworm host strain, we successfully increased the amount of rMmIL-4 by six-fold. Thus, seeking an optimal strain to produce the target recombinant proteins effectively would be a short cut to enhance the productivity in the silkworm-BEVS. Besides, our results of analysis using a variety of silkworm strains provide insights into the exploring of the potential baculovirus-sensitive genes for breeding better optimal strains for mass-production of recombinant proteins.

Acknowledgment We thank Dr. Imanishi (National Institute of Agrobiological Sciences, Japan) for kindly providing the NIAS-Bm-oyanagi2 (BmO2) cell line. This work was supported by the Japan Science and Technology Agency (JST) for the Program for Creating Start-ups from Advanced Research and Technology (START Program). Conflict of interest The authors declare no conflict of interest.

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Fig. 1. (A) Construction of transfer plasmids to generate recombinant baculoviruses. The transfer plasmid was under the control of the polyhedrin promoter (polh) and followed by an SV40 polyadenylation signal (SV40 pA). B1 and B2: recombination sites for Gateway cloning, L21: leader sequence for enhancing translation efficiency, 30K SP: signal peptide of silkworm 30 kDa protein, MmIL-4: Murine Interleukin-4, H8: 8x Histidine tag, STREP: Strep-tag, H6: 6x Histidine tag, TEV: tobacco etch virus protease cleavage site. (B) Time-course expression analysis of rMmIL-4 in the media of BmO2 cells and sera of silkworm larvae infected with the recombinant BmNPV. The culture medium and serum were harvested at 3, 4, and 5 days post-infection (dpi). All samples were resolved on a 15% SDS-PAGE. The rMmIL-4 protein was also detected

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by Western blotting using HisProbe-HRP Antibody. The arrow indicates the expression of rMmIL-4 Fig. 2. Purification of N-tagged (A) and C-tagged (B) rMmIL-4 by Nickel (left panel) and Strep-Tactin (right panel) affinity chromatography. M: molecular mass markers, IP: input, FT: flow-through, WS: wash fraction, lanes 1–4 (left panel): elution fractions by 100 mM imidazole, lanes 5–9 (left panel): elution fractions by 500 mM imidazole. Elution fractions (lane 2–9) were concentrated and diluted with PBS (lane E) and further purified by Strep-Tactin affinity chromatography. Lanes 1-7 (right panel): elution fractions by 2.5mM desthiobiotin. All samples were visualized by Coomassie Brilliant Blue (CBB) R-250 staining. The arrows indicate the expressions of rMmIL-4. Fig. 3. Biological activity of rMmIL-4 by the proliferation assay using HT-2 cells. The activities of N- and C-tagged rMmIL-4 were measured and compared with commercial rMmIL-4. The data were represented by the means ± standard error of five values obtained independently. Fig. 4. Screening of silkworm strains for mass-production of rMmIL-4. (A) Sera (1μl apply) from each silkworm infected with recombinant baculoviruses encoding C-tagged rMmIL-4 were resolved in a 15% SDS-PAGE and rMmIL-4 was detected by Western blotting using HisProbe-HRP. Three individual samples were prepared from each strain, and d17 sera were used as a control. The arrows indicate the expressions of rMmIL-4. (B) After two-step purification, purified rMmIL-4 from four strains, d17, e10, n22, and

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n70, which showed high secretion efficiency, were compared and analyzed on a 15% SDS-PAGE. The arrows indicate the expressions of rMmIL-4. (C) Comparison of the yield after purification of rMmIL-4 recovered from 10 ml of each strain serum. All samples of rMmIL-4 proteins obtained from (B) were quantified by YabGelImage software using BSA as a standard.

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