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A fibroin secretion-deficient silkworm mutant, Nd-sD, provides an efficient system for producing recombinant proteins
ARTICLE IN PRESS Insect Biochemistry and Molecular Biology Insect Biochemistry and Molecular Biology 35 (2005) 51–59 www.elsevier.com/locate/ibmb
A fibroin secretion-deficient silkworm mutant, Nd-sD, provides an efficient system for producing recombinant proteins Satoshi Inouea,1, Toshio Kandaa, Morikazu Imamuraa,2, Guo-Xing Quana,3, Katsura Kojimaa, Hiromitsu Tanakaa, Masahiro Tomitab, Rika Hinob, Katsutoshi Yoshizatob, Shigeki Mizunoc, Toshiki Tamuraa, a Insect Biotechnology and Sericology Department, National Institute of Agrobiological Sciences, 1-2 Owashi, Tsukuba, Ibaraki 305-8634, Japan Hiroshima Tissue Regeneration Project, Hiroshima Prefecture Collaboration of Regional Entities for Advancement of Technological Excellence, Japan Science and Technology Corporation, 3-10-32 Kagamiyama, Higashihiroshima, Hiroshima 739-0046, Japan c Department of Agricultural and Biological Chemistry, College of Bioresource Sciences, Nihon University, 1866 Kameino, Fujisawa 252-8510, Japan
b
Received 3 September 2004; received in revised form 12 October 2004; accepted 15 October 2004
Abstract The silkworm Nd-sD mutant is silk fibroin-secretion deficient. In the mutant, a disulfide linkage between the heavy (H) and light (L) chains, which is essential for the intracellular transport and secretion of fibroin, is not formed because of a partial deletion of the L-chain gene. To utilize the inactivity of the mutant L-chain, we investigated the possibility of using the Nd-sD mutant for the efficient production of recombinant proteins in the silkworm. A germ line transformation of the mutant with a normal L-chain-GFP fusion gene was performed. In the transgenic mutant, normal development of the posterior silk gland (PSG) was restored and it formed a normal cocoon. The biochemical analysis showed that the transgenic silkworms expressed the introduced gene in PSG cells, produced a large amount of the recombinant protein, secreted it into the PSG lumen, and used it to construct the cocoon. The molar ratio of silk proteins, H-chain:L-chain-GFP:fibrohexamerin, in the lumen and cocoon in the transgenic silkworm was 6:6:1, and the final product of the fusion gene formed about 10% of the cocoon silk. This indicates that the transgenic mutant silkworm possesses the capacity to produce and secrete the recombinant proteins in a molar ratio equal to that of the fibroin H-chain, contributing around half molecules of the total PSG silk proteins. r 2004 Elsevier Ltd. All rights reserved. Keywords: Intracellular transport; Transgenic; Recombinant protein; Bioreactor; PiggyBac; Fibroin; Silkworm; Bombyx; Nd-s mutant
1. Introduction The use of a transgenic silkworm as a bioreactor for the production of recombinant proteins has been realized with the success of a germ line transformation Corresponding author. Tel./fax: +81 298 38 6091.
E-mail address: [email protected] (T. Tamura). Present address: MRC Toxicology Unit, University of Leicester, Leicester LE1 9HN, UK. 2 Present address: Prion Disease Research Center, National Institute of Animal Health, Tsukuba, Ibaraki 305-0856, Japan. 3 Present address: Molecular Entomology, Great Lakes Forestry Centre, Canadian Forest Service, Marie, Ontario, Canada P6A 5M7. 1
0965-1748/$ - see front matter r 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.ibmb.2004.10.002
method using transposon piggyBac (Tamura et al., 2000). The domesticated silkworm, Bombyx mori, possesses efficient protein production organs called silk glands, for making silk proteins. In this insect, more than half of the dry weight of the cocoon consists of silk proteins. Therefore, more than half of the nutrients consumed by the larvae are converted into silk proteins. Recently, it was reported that recombinant protein could be produced in the silk gland of transgenic silkworms (Tomita et al., 2003). However, the production was not efficient. The major components of the cocoon of the transgenic silkworm consisted of the products of the native silk genes, suggesting that one of
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the critical factors for the production of recombinant protein is related to the system of silk protein secretion. The major components of cocoon silk in nontransgenic normal silkworms are fibroin and sericin. Fibroin is synthesized in the posterior silk gland (PSG) and accumulates in the lumen of the middle silk gland (MSG), where sericin is synthesized. Then, the fibroin is secreted into the cocoon via the anterior silk gland (ASG). The silk fibroin contains three polypeptides: a 350-kDa heavy chain (H-chain, Shimura, 1983), a 26kDa light chain (L-chain, Yamaguchi et al., 1989), and fibrohexamerin (fhx), originally named P25(Inoue et al., 2000). In the PSG, these proteins form a large protein complex called an elementary unit of fibroin, which consists of a 6:6:1 molar ratio of the H-chain:Lchain:fibrohexamerin (Inoue et al., 2000). The L-chain is linked with the H-chain by a disulfide bond between Cys-172 of the L-chain and Cys-c20 (twentieth residue from the carboxy terminus) of the H-chain (Tanaka et al., 1999b). One molecule of fibrohexamerin interacts non-covalently with six sets of H-L heterodimers to form the elementary unit in the endoplasmic reticulum (ER) (Inoue et al., 2004), and the three N-linked oligosaccharide chains of fibrohexamerin contribute to maintaining the integrity of the elementary unit (Inoue et al., 2000). This molecular complex is essential for quality control in the ER (Reddy and Corley, 1998; Ellgaard et al., 1999; Fassio and Sitia, 2002), enabling the efficient intracellular transport and secretion of significant amounts of fibroin into the lumen (Inoue et al., 2004). The Nd-sD mutant is characterized by an immature PSG and less than 1% of the normal secretion level of fibroin, which leads to the production of a very thin, naked-pupa cocoon that consists mostly of sericin. The gene is mapped to the L-chain gene (fib-L) locus on chromosome 14 (Takei et al., 1984a). The mutation is caused by a deletion downstream from exon III, which causes the recombination of sequences in the third intron with sequences in the far downstream region. This recombination created a chimeric gene containing the first three exons of fib-L and two new exons, IV0 and V0 , from the far downstream region. This mutant chimeric Nd-sD L-chain lacks Cys-172, which was encoded by the original sixth exon, and consequently cannot form a disulfide linkage with the H-chain (Mori et al., 1995). The H–L disulfide linkage contributes to the efficient intracellular transport and secretion of silk fibroin, which is analogous to IgM production (Sitia et al., 1990), and the accumulation of L-chain-deficient fibroin in the ER might inhibit the development of PSG cells. It has been reported that the normal L-chain introduced into the Nd-sD mutant is secreted in the lumen and this suggests that the mutant might be used to increase the production and secretion of L-chainfused recombinant protein.
In this study, the L-chain-GFP fusion gene was introduced into the Nd-sD mutant to assess its ability to produce recombinant protein. The L-chain– GFP fusion gene was expressed in the PSG of the mutant transgenic silkworm; large amounts of recombinant protein were secreted into the lumen and spun into the cocoon. The Nd-sD phenotype, which consisted of an immature PSG and thin cocoon, was rescued dramatically and the cocoon was almost indistinguishable from those of normal silkworms. A single cocoon contained about 8.6 mg dry weight (16.6 mmol) of L-chain-GFP fusion protein, which corresponds to about 10% of the total cocoon silk protein. The molar ratio of H-chain:Lchain-GFP:fibrohexamerin in the secreted fibroin was strictly 6:6:1, indicating that the formation of complexes of the three proteins is critical, even with the fusion protein.
2. Materials and methods 2.1. Silkworms Fertilized eggs of fibroin-secreting wild-type B. mori C108 and the fibroin-secretion-deficient, naked-pupa mutant of B. mori Nd-sD (with a mutation of the fibroin L-chain gene, fib-L) (Takei et al., 1984a; Mori et al., 1995) were supplied by the Insect Genetics Laboratory of the National Institute of Agrobiological Sciences, Japan. The larvae were reared on an artificial diet (Nihon Nosan) at 25 1C. 2.2. Plasmid vectors The helper plasmid vector, pHA3PIG (6.2 kb), is described elsewhere (Tamura et al., 2000). The DsRed2 cDNA that was excised from the pDsRed2-1 vector (Clontech) was used to replace EGFP cDNA in pBac(3 P3-EGFPafm) (Horn and Wimmer, 2000), giving rise to pBac(3 P3-DsRed2). The L-chain 50 flanking region (from 600 to +34) (Kikuchi et al., 1992), the L-chain cDNA sequence (from +34 to +767) [2], and the L-chain 30 -flanking region (from +13114 to +13597) (Kikuchi et al., 1992) were amplified by PCR from genomic DNA prepared from B. mori. EGFP cDNA was excised from the pEGFP vector (Clontech). All of the cDNA fragments were inserted into pBac(3xP3-DsRed2) to produce pBac(3xP3DsRed2+L-chain-GFP) (Fig. 1A). 2.3. Transformation Plasmid DNA was purified using the QIAGEN Midi (Qiagen). To break embryonic diapause, eggs were treated within 4 h after oviposition with 20% HCl for 1 h at 25 1C. A mixture of helper plasmid pHA3PIG
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2.5. Northern blot analysis Total RNA from PSG or MSG at the 3rd day of the 5th instar was purified using ISOGEN (Nippongene). Total RNA was subjected to Northern blotting and hybridized with the probe as follows. Part of the L-chain cDNA sequence (+1 to +357; corresponding to exons I to III) that is common to both the normal and Nd-sD Lchains was amplified from pFL18 by PCR (Yamaguchi et al., 1989). Part of the GFP cDNA sequence (+1 to +357) was amplified from pEGFP (Clontech) by PCR. Part of the B. mori elongation factor a-I isoform cDNA sequence (from +1 to +1682) was amplified from pBmEF-1 a-1 by PCR (Kamiie et al., 1993). Fig. 1. Restriction enzyme map of a piggyBac vector and genomic DNA of the normal breed (C108) and Nd-sD. (A) Design of a piggyBac vector for L-chain-GFP expression in PSG cells. The DsRed2 marker gene construct is placed immediately downstream from the Pax-6 artificial promoter sequences (3 P3), enabling its expression in photoreceptor cells. The L-chain promoter is fused to the L-chainGFP coding sequence and L-chain 30 -untranslated sequences. Inserted terminal repeats of piggyBac transposase (arrows) and EcoRI sites (E) are indicated. (B) Physical maps of the normal (C108) and Nd-sD mutant L-chain genes. The normal gene (Fib-L) contains seven exons (exon I-VII), whereas the chimeric mutant gene contains exons I–III of Fib-L and exons IV0 and V0 derived from the far downstream region.
2.6. Protein analysis Preparation of fibroin samples from PSG lumen, PSG tissues, and cocoons has been described elsewhere (Tanaka et al., 1999a; Inoue et al., 2000). Western blotting of fibroin samples was carried out, as described previously (Inoue et al., 2000), with the following antibodies: rabbit anti-H-chain or anti-L-chain polyclonal antibody (Inoue et al., 2000), mouse anti-fhx polyclonal antibody (Inoue et al., 2000), or rabbit antiGFP (Invitrogen) antibodies. 2.7. Purification of L-chain-GFP
(2–3 nl) [11] and vector DNA (0.2 mg/ml of each) in 0.5 mM phosphate buffer (pH 7.0) with 5 mM KCl was injected into 476 eggs 4 h after the acid treatment. Injected eggs were placed in a humidified box at 25 1C until hatching. Hatched larvae were transferred to an artificial diet and grown to adulthood. G0 moths were mated within the same family, and 7-day G1 embryos were screened for DsRed2 expression in the stemmata and nervous system tissues (Thomas et al., 2002) with a Leika MZFL III fluorescence microscope (Leika) equipped with filter sets for DsRed. DsRed2 expression in screened silkworms was confirmed in the compound eyes of G1 moths. Moths expressing DsRed2 were mated with each other. The G2 larvae that were most strongly positive for DsRed2 were used as transgenic lines. Green fluorescent protein in the dissected silk gland and cocoon was observed with a Leika MZFL III fluorescence microscope (Leika) equipped with filter sets for GFP2.
To purify the L-chain-GFP fusion protein, a fibroin sample from the PSG lumen was subjected to 0.1% SDS, 12.5% PAGE (SDS-PAGE); the L-chain-GFP fusion protein was electro-eluted in 5 mM Tris, 38.4 mM glycine. The purity of the L-chain-GFP fusion protein was judged by silver staining and Western blotting. 2.8. Quantitative ELISA Standard curves for the H-chain, L-chain, and fibrohexamerin are described elsewhere (Inoue et al., 2000). Standard curves for L-chain-GFP were made using the purified L-chain-GFP protein; the protein was dissolved in 20 mM Tris–HCl (pH 8.0), 2 M urea, and subjected to ELISA using rabbit anti-L-chain antibody. Protein samples prepared from the PSG lumen or the cocoon shell were dissolved in 20 mM Tris–HCl (pH 8.0), 2 M urea, and subjected to quantitative ELISA as described elsewhere [3].
2.4. Southern blot analysis Genomic DNA was extracted from G2 moths (Tamura et al., 2000). EcoRI-digested genomic DNA (5 mg) was subjected to Southern blotting and hybridized with the L-chain 30 –flanking region (from +887 to +1354) from pFL18 (Yamaguchi et al., 1989) as a probe for the normal L-chain gene.
3. Results 3.1. Construction of Nd-sD mutant transgenic silkworms We constructed the transformation vector pBac(3 P3-DsRed2+L-chain-GFP) (Fig. 1A) and
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injected it with helper plasmid into 476 eggs of Nd-sD at the preblastodermal stage. Fifteen transgenic silkworms were verified by screening for DsRed2 emission in the stemmata and nervous tissues of embryos or moths (Fig. 2B), as reported by Thomas et al. (2002) in the transgenic line containing the 3XP3GFP insert, and even in a silkworm with normal brown color stemmata
and compound eyes. These silkworms were mated with each other to produce the LGL4 5 line, which was characterized by a strong DsRed2 emission in the stemma and complex eyes. To confirm the insertion of the fusion gene and to identify the number of integrated DNA molecules in the Nd-sD genome of the transgenic line, the EcoRI-digested genomic DNA of wild type, NdsD, and the LGL4 5 line were investigated by Southern blot hybridization with a normal L-chain-specific probe (Fig. 1B). The probe hybridized with one specific 8.7-kb band in wild-type silkworms (Fig. 2B lane 1), no band in Nd-sD mutants (Fig. 2B lane 2), as expected, and 9.8-, 7.8-, 5.6-, and 4.1-kb bands in the LGL4 5 line (Fig. 2B lane 3). This indicated that the LGL4 5 line was a transgenic line containing the normal L-chain-GFP fusion gene in at least four different loci of the original Nd-sD genome. 3.2. Restoration of PSG development and fibroin secretion in the transgenic line The most prominent phenotypic features of the Nd-sD mutant silkworm are an immature PSG (Fig. 3A(b)) and the production of a very thin cocoon (Fig. 3B(b)) relative to that of a normal silkworm (Figs. 3A(a), B(a)). The phenotypes of the transgenic line LGL4 5 were dramatically changed from those of Nd-sD with respect to the extensive development of the PSG (Fig. 3A(c)) and the production of a thick cocoon (Fig. 3B(c)) similar to that of the normal C108 (Figs. 3A(a), B(a)). The mean weight of cocoons produced by transgenic line LGL4 5 was 78.4 mg (n ¼ 50), which was much heavier than that of Nd-sD (17.7 mg; n ¼ 50), but less than half that of the normal C108 (187 mg; n ¼ 50). These results indicate that the prominent phenotypes of the Nd-sD mutant can be largely converted to those of the normal fibroin-producing silkworm by transgenesis with the normal L-chain-GFP fusion gene. 3.3. Expression and secretion of functional L-chain– GFP fusion protein in transgenic silkworms
Fig. 2. Morphological characters of transgenic Nd-sD mutant inserted using the 3XP3DsRed2 marker gene with genomic Southern blotting. (A) Expression of DsRed2 in the compound eyes of G1 moths (upper) and in the stemmata of a 7-day-old G2 embryo (lower). Note that the mutant possesses black stemmata and compound eyes. The marker is still useful for detecting transgenic silkworm. Left panel; Nd-sD, right panel; transgenic line. An arrowhead and arrows point to the ocelli and abdominal nervous system, respectively. Scale bars: 0.5 mm. (B) Southern blot of EcoRI-digested genomic DNA (5 mg each) from C108 (lane 1), Nd-sD (lane 2), and the transgenic line (lane 3) hybridized with the normal L-chain specific probe, as shown in Fig. 1B.
The emission of green fluorescence due to the expression of the integrated GFP gene was observed in the transgenic silkworm at the 5th day of the 5th instar, when the secretion of fibroin reached the maximal level, in the PSG cells and the lumen of whole silk glands (from PSG, MSG to ASG) (Fig. 3A(c) and (c*)). The fluororescence was not detected in other tissues at whole stages, suggesting that the fusion gene specifically expressed in PSG cells. Green fluorescence was also detected in the entire cocoon produced by the transgenic silkworm (Fig. 3B(c)). These observations suggested that the L-chain-GFP fusion protein was expressed from the integrated fusion gene in the transgenic silkworm.
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Fig. 3. GFP fluorescence in the PSG and cocoon of the transgenic line. (A) A pair of silk glands at the 5th day of the 5th instar larvae of C108 (a), Nd-sD (b), and the transgenic line (c) were observed under bright field (bright) and with the GFP fluorescence system (GFP). The ASG, MSG, and PSG of the transgenic line were observed under higher magnification with the EGFP fluorescence system (c*). ASG: anterior silk glands, MSG: middle silk glands, PSG; posterior silk glands. (B) Cocoons produced by C108 (a), Nd-sD (b), and the transgenic line (c) were observed similarly.
In order to prove this, Northern blots of total RNA preparations from PSG or MSG cells of the normal breed C108, Nd-sD, or the transgenic line LGL4 5 were probed with cDNA for the L-chain sequence derived from exons I, II, and III (common to C108 and Nd-sD Lchains) or cDNA for GFP. The L-chain cDNA probe hybridized to 1.3- and 1.1-kb bands in the RNA preparations from the PSGs of C108 and Nd-sD, respectively (Fig. 4A, left panel, lanes 1 and 2). The amount of Nd-sD L-chain mRNA was less than 5% of that in C108. The L-chain cDNA probe did not hybridize to RNA preparations from the MSG of either C108 or Nd-sD (Fig. 4A, left panel, lanes 4 and 5). These results are consistent with those reported previously (Takei et al., 1987).
Fig. 4. Detection of L-chain-GFP transcript and protein in the transgenic line. (A) Northern blots of total RNA (5 mg/lane) from the PSG (lanes 1–3) and MSG (lanes 4–6) of C108 (lanes 1 and 4), Nd-sD (lanes 2 and 5), and the transgenic line (lanes 3 and 6) hybridized with the L-chain cDNA probe (exons I–III, 357 bp) common to both normal and Nd-sD L-chain mRNA, GFP cDNA probe (357 bp), or the cDNA probe for the B. mori elongation factor a-I isoform. (B) Western blot of cocoon proteins of C108 (a), Nd-sD (b), and the transgenic line (c). The PSG tissue (lanes 1 and 3) and the cocoon (lanes 2 and 4) proteins were separated by SDS-PAGE without reduction (lanes 1 and 2) or under reducing conditions (lanes 3 and 4) and subjected to Western blotting with anti-H-chain (left), anti-L-chain (middle), or anti-GFP antibodies (right).
The L-chain cDNA probe hybridized to 2.1- and 1.1kb components in the RNA preparation from the PSG of transgenic line LGL4 5 (Fig. 4A, left panel, lane 3).
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The 2.1-kb, but not the 1.3-kb, component hybridized with the GFP cDNA probe (Fig. 4A, right panel, lane 3). The hybridizing RNA bands were interpreted as normal L-chain mRNA (1.3 kb), Nd-sD L-chain mRNA (1.1 kb), and normal L-chain-GFP fusion gene mRNA (2.1 kb). Judging from the relatively strong intensity of the 2.1-kb band, it appeared that the normal L-chainGFP fusion transgenes were actively transcribed in a PSG-specific manner in the transgenic line LGL4 5. To detect L-chain or L-chain-GFP fusion polypeptides, proteins from PSG tissue or from cocoon shells of C108, Nd-sD, or LGL4 5 were subjected to Western blotting using anti-H-chain, anti-L-chain, or anti-GFP antibodies, before and after the reductive cleavage of disulfide bonds. In the normal C108 strain, the L-chain was detected in two forms, either unbound or bound to H-chain, in the PSG tissue sample (Fig. 4B(a), lanes 1 and 3), but only the H-chain-bound form was detected in the cocoon sample (Fig. 4B(a), lanes 2 and 4), as reported (Takei et al., 1987). In Nd-sD, the mutant Lchain was detected in an unbound form in the PSG tissue sample (Fig. 4B(b), lanes 1 and 3), but not in the cocoon sample (Fig. 4B(b), lanes 2 and 4), as reported (Takei et al., 1987). In the transgenic line, the anti-Lchain antibody reacted with 52.5- and 27-kDa bands in the PSG tissue sample after reducing the disulfide bonds (Fig. 4B(c), middle panel, lane 3), whereas the anti-GFP antibody reacted with only the 52.5-kDa band under the same conditions (Fig. 4B(c), right panel, lane 3). These results strongly suggest that the 52.5-kDa band is the normal L-chain-GFP fusion protein and that the 27kDa band is the Nd-sD mutant L-chain. As expected, only the normal L-chain-GFP fusion protein was detected in the cocoon protein sample in the reactions with anti-L-chain antibody or anti-GFP antibody (Fig. 4B(c), middle and right panels, lane 4). The normal Lchain-GFP fusion protein was associated with the Hchain before reduction of the disulfide bonds, as shown by the reaction with both anti-L-chain and anti-GFP antibodies (Fig. 4, middle and right panels, lane 2). These results suggest that the normal L-chain-GFP fusion genes, which were integrated into the genome of the transgenic silkworm, are expressed as proteins that behave like the normal L-chain and contribute to the restoration of the phenotype with respect to high secretion levels of fibroin and the production of nearly normal cocoons. 3.4. Quantification of the normal L-chain-GFP fusion protein in cocoons The molar ratio of H-chain:L-chain:fibrohexamerin in the elementary unit of fibroin produced by the normallevel fibroin-producing breed of silkworms is 6:6:1 (Inoue et al., 2000). We used quantitative ELISA to determine whether the normal L-chain-GFP fusion
protein expressed in the transgenic silkworm participated in the formation of an elementary unit with a similar molar ratio. For this purpose, the normal L-chain-GFP fusion protein was purified from the fibroin secreted into the PSG lumen of the transgenic silkworm, as described in the Methods and materials. The purity of the protein sample was examined by SDS-PAGE, followed by silver staining or Western blotting. It behaved as a single 52.5kDa band (Fig. 5A(a)) and reacted with the anti-L-chain or anti-GFP antibody, but not with the anti-H-chain or anti-fibrohexamerin antibody (Fig. 5A(b)–(e)). This protein sample was then used to make an ELISA standard curve for the L-chain-GFP fusion protein (Fig. 5B). ELISA standard curves for H-chain and fibrohexamerin were made, as described (Inoue et al., 2000). The results of the quantitative ELISA for cocoon protein samples are summarized in Table 1. The molar ratio of H-chain:L-chain:fibrohexamerin was close to 6:6:1 for the normal breed C108, 6:0:1 for Nd-sD, and close to 6:6:1 for the transgenic line LGL4 5. These results
Fig. 5. Purification of L-chain-GFP and the production of a standard curve for quantitative ELISA. (A) Purification of L-chain-GFP. A fibroin sample (lane 1) from the transgenic line was separated by SDSPAGE without cleaving the disulfide bonds, and L-chain-GFP (lane 2) was recovered by electro-elution. The purity was judged by silver staining (a), and Western blotting using anti-H-chain (b), anti-L-chain (c), anti-fibrohexamerin (d), and anti-GFP (e) antibodies. (B) ELISA for the purified L-chain-GFP. The purified L-chain-GFP was subjected to ELISA using anti-L-chain antibody. The reactivity was monitored by the reaction with horseradish peroxidase-conjugated antirabbit IgG followed by the color-developing reaction (A490) with o-phenylenediamine dihydrochloride.
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2.23 104 ND 2.14 104 2.21 104 70 2.17 104 5.75 103 ND 1.1 104 C108 Nd-sD Transgenic line
0.517 0.401 0.622
0.354 ND 0.274
0.196 0.421 0.18
6.31 104. 245 7.58 104
1.12 103 3 1.04 103
H-chain L-chain or L-chain-GFP H-chain L-chain or L-chain-GFP
Fhx H-chain
fhx
pmol ng/protein (100 mg) A490 a
Breed
Table 1 Molar ratios of H-chain, L-chain or L-chain-GFP, and fhx in the fibroin of cocoons
We demonstrated that the Nd-sD mutant, which possesses degenerated PSG and expels only sericin in the cocoon, is useful for producing a large amount of Lchain fused recombinant proteins in the transgenic silkworm. The amount of the L-chain-GFP fusion proteins in our experiment exceeded 10% of the proteins in the cocoon when the gene was introduced into the mutant. In addition, the Nd-sD mutant was rescued by the introduction of L-chain-GFP fusion protein gene, suggesting that the protein production and secretion machinery of the mutant PSG had recovered. We used the fusion gene with a promoter from the 600-bp upstream region of the L-chain gene. The expression of the inserted fusion gene was apparently lower than that in the normal type assessed by Northern blotting (Fig. 4A compare lanes 1 and 3). However, the tissue-specificity of the inserted gene was maintained in the transgenic silkworm, indicating that signals related to the tissue-specific expression of the L-chain gene are localized to this region. There are two possible explanations for the low level of gene expression: (i) the inserted gene disturbed a critical chromatin structure at the insertion position, which affected its expression, or (ii) the binding sites of some transcriptional activators were not present in the promoter region used. For the latter possibility, using a longer upstream region as the promoter may increase the mRNA level. The fibroin H- and L-chain genes and the fibrohexamerin gene share the same transcriptional regulation system, and are expressed in a coordinated manner in the PSG (Bello et al., 1994), despite the fact that the three genes are located on different chromosomes: FibH is on chromosome 25, fib-L is on chromosome 14, and fibrohexamerin is on chromosome 2. Otherwise, it might be better to use the promoter of H-chain, or fibrohexamerin genes for the L-chain, or the targeted gene expression system of GAL4/UAS (Imamura et al., 2003). Reduced expression of the introduced gene, as compared to the endogenous gene, is also observed when the promoters of other genes are used (Suzuki et al., 2003). Another important factor is the secretion of the recombinant proteins in PSG. The ER provides an
L-chain or L-chain-GFP
4. Discussion
57 n ¼ 6 times; 7, SD; ND, not detected. a Values obtains by ELISA. Samples (all in 100 ml) assayed were 10 ng (for H-chain), 50 ng (for L-chain) and125 ng (for fhx) proteins from the cocoons of C108 and the transgenic line; 2 mg (for Hchain), 10 mg for L-chain, 100 mg for fhx proteins from Nd-sD.
5.9270.04:5.9870.05:1 6.3170.07:—:1 6.2070.07:6.1770.03:1 3.73 103 11.1 3.47 103
fhx
Molar ratio
suggest that the L-chain-GFP fusion proteins expressed in the transgenic silkworm participated in the assembly of an elementary unit of fibroin whose molecular constitution was similar to that formed in the normal breed, irrespective of the presence of Nd-sD mutant Lchain proteins in the PSG cells. The content of L-chainGFP fusion protein was calculated to be about 110 mg (212 nmol)/mg of the cocoon protein or about 8.6 mg (16.6 mmol) per dried cocoon (78.4 mg) produced by the transgenic silkworm.
H-chain : L-chain or L-chain-GFP : fhx
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environment and mechanisms to facilitate the proper formation of newly synthesized proteins into higher order conformations. The quality control function of the ER (Hammond and Helenius, 1995) is critical to ensuring that only properly folded proteins enter secretory pathways. In the case of silk fibroin, a disulfide linkage between the H- and L-chains (Takei et al., 1984b, 1987), the addition of three N-linked oligosaccharide chains onto fibrohexamerin (Tanaka et al., 1999a; Inoue et al., 2000), and the assembly of the elementary unit (Inoue et al., 2000) all take place in the ER (Inoue et al., 2004). These processes are important for the efficient secretion of fibroin. The significance of the H–L linkage for the efficient secretion of fibroin has been suggested by genetic and biochemical studies of the fibroin secretion deficient Nd-sD mutant (Takei et al., 1987; Mori et al., 1995; Tanaka et al., 1999b). The application of the transgenic silkworm as a bioreactor to produce recombinant proteins has several advantages: (i) the cost of silkworm rearing is much lower than the cost of rearing other animals; (ii) large quantities of recombinant protein can be produced (0.5–2 mmol fibroin per dried 200–500 mg cocoon); (iii) post-translational modifications are similar to those of mammalian proteins, except for oligosaccharide processing; (iv) the protein composition is relatively simple; and (v) the risk of disease is lower. Recently, Tomita et al. (2003) constructed a wild-type transgenic silkworm containing the L-chain-GFP and Lchain-human type III procollagen genes; the exogenously expressed L-chain-GFP protein was secreted and was found in the dried cocoon. However, the secretion efficiency of the fusion proteins in the wild-type transgenic silkworm was very low. By contrast, the Nd-sD mutant transgenic silkworm we established showed much higher production of the recombinant protein. The reason for this higher production by the mutant is outlined in Fig. 6. The fusion protein produced in the normal breed competes with the normal L-chain in the process of formation of the H–L linkage. The endogenous normal L-chain has a greater affinity for the H-chain than does the L-chain-GFP fusion protein. Therefore, the wild-type transgenic silkworm secreted relatively more endogenous normal L-chain than the fusion protein. Actually, the molar ratio of endogenous L-chain to L-chain-GFP fusion protein in the cocoon of the transgenic normal breed is about 10:1, concomitant with the accumulation of a relatively large amount of fusion protein in PSG cells (S. Inoue et al., unpublished data). However, the mutant L-chain in the transgenic Nd-sD mutant cannot compete with the fusion protein. Therefore, only the fusion protein is secreted into the lumen. Indeed, the fusion protein was secreted efficiently. In this study, a cocoon of the mutant contained about 8.6 mg (16.6 mmol) of the functional (green fluorescence emitting) L-chain-GFP fusion pro-
Fig. 6. Model of the intracellular transport of recombinant proteins produced in the transgenic silkworms. H, fibroin H-chain; L, fibroin Lchain; Lm, fibroin L-chain produced by Nd-sD mutant; fhx, fibrohexamerin. The L-chain-GFP fusion protein (L+GFP) produced in the PSG cells of the normal breed has lower affinity in the formation of an S–S linkage with the H-chain, compared with the normal Lchain. Therefore, less of the fusion protein is secreted in the normal breed. However, the fusion protein in the mutant does not compete with the mutant L-chain. Therefore, only the fusion protein linked with H-chain is secreted in the mutant.
tein (Table 1). The efficient secretion of L-chain-GFP fusion protein provides the Nd-sD mutant transgenic line with the advantage of higher protein yields than in the wild-type transgenic silkworm. In addition, the L-chainGFP fusion protein is more easily purified from cocoons of the Nd-sD mutant transgenic line, because the cocoons lack the endogenous L-chain. Our future work will focus on designing other functional proteins and establishing an easy purification system with high yields of the recombinant proteins from cocoons. The tandem affinity purification (TAP) system may prove to be a powerful tool for the purification of functional foreign recombinant proteins from cocoons (Rigaut et al., 1999).
Acknowledgments We would like to thank Dr. Ernst A. Wimmer of Universitat Bayeyth for kindly providing pBac(3 P3EGFPafm), Kazuko Seo and Hiroko Yamazaki for technical assistance, and Dr. Michelle A. Hughes (University of Leicester) for a critical reading of the manuscript. This work was supported by the Ministry of Agriculture, Forest, and Fisheries and by the Program for the Promotion of Basic Research Activities for Innovative Bioscience, Japan.
References Bello, B., Horard, B., Couble, P., 1994. The selective expression of silk–protein-encoding genes in Bombyx mori silk gland. Bull. Inst. Pasteur 92, 81–100. Ellgaard, L., Molinari, M., Helenius, A., 1999. Setting the standards: quality control in the secretory pathway. Science 286, 1882–1888.
ARTICLE IN PRESS S. Inoue et al. / Insect Biochemistry and Molecular Biology 35 (2005) 51–59 Fassio, A., Sitia, R., 2002. Formation, isomerisation and reduction of disulphide bonds during protein quality control in the endoplasmic reticulum. Histochem. Cell Biol. 117, 151–157. Hammond, C., Helenius, A., 1995. Quality control in the secretory pathway. Curr. Opin. Cell Biol. 7, 523–529. Horn, C., Wimmer, E.A., 2000. A versatile vector set for animal transgenesis. Dev. Genes Evol. 210, 630–637. Imamura, M., Nakai, J., Inoue, S., Quan, G.-X., Kanda, T., Tamura, T., 2003. Targeted gene expression using the Gal4/UAS system in the silkworm Bombyx mori. Genetics 165, 1329–1340. Inoue, S., Tanaka, K., Arisaka, F., Kimura, S., Ohtomo, K., Mizuno, S., 2000. Silk fibroin of Bombyx mori is secreted, assembling a high molecular mass elementary unit consisting of H-chain, L-chain, and P25, with a 6:6:1 molar ratio. J. Biol. Chem. 275, 40517–40528. Inoue, S., Tanaka, K., Tanaka, H., Ohtomo, K., Kanda, T., Imamura, M., Quan, G-X., Kojima, K., Yamashita, T., Nakajima, T., Taira, H., Tamura, T., Mizuno, S., 2004. Assembly of the silk fibroin elementary unit in endoplasmic reticulum and a role of L-chain for protection of a-1,2-mannose residues in N-linked oligosaccharide chains of fibrohexamerin/P25. Eur. J. Biochem. 271, 1–11. Kamiie, K., Taira, H., Ooura, H., Kakuta, A., Matsumoto, S., Ejiri, S., Katsumata, T., 1993. Nucleotide sequence of the cDNA encoding silk gland elongation factor 1 alpha. Nucleic Acids Res 21, 742. Kikuchi, Y., Mori, K., Suzuki, S., Yamaguchi, K., Mizuno, S., 1992. Structure of the Bombyx mori fibroin light-chain-encoding gene: upstream sequence elements common to the light and heavy chain. Gene 110, 151–158. Mori, K., Tanaka, K., Kikuchi, Y., Waga, M., Waga, S., Mizuno, S., 1995. Production of a chimeric fibroin light-chain polypeptide in a fibroin secretion-deficient naked pupa mutant of the silkworm Bombyx mori. J. Mol. Biol. 251, 217–228. Reddy, P.S., Corley, R.B., 1998. Assembly, sorting, and exit of oligomeric proteins from the endoplasmic reticulum. Bioessays 20, 546–554. Rigaut, G., Shevchenko, A., Rutz, B., Wilm, M., Mann, M., Seraphin, B., 1999. A generic protein purification method for protein complex characterization and proteome exploration. Nat. Biotechnol. 17, 1030–1032. Shimura, K., 1983. Chemical composition and biosynthesis of silk proteins. Experimentia 39, 455–461. Sitia, R., Neuberger, M., Alberini, C., Bet, P., Fra, A., Valetti, G., Williams, G., Milstein, C., 1990. Developmental regulation of IgM
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secretion: the role of the carboxy-terminal cysteine. Cell 60, 781–790. Suzuki, M.G., Funaguma, S., Kanda, T., Tamura, T., Shimada, T., 2003. Analysis of the biological functions of a doublesex homologue in Bombyx mori. Dev. Genes 213, 345–354. Takei, F., Kimura, K., Mizuno, S., Yamamoto, T., Shimura, K., 1984a. Genetic analysis of the Nd-s mutation in the silkworm. Jpn. J. Genet. 59, 307–313. Takei, F., Oyama, F., Kimura, K., Hyodo, A., Mizuno, S., Shimura, K., 1984b. Reduced level of secretion and absence of subunit combination for the fibroin synthesized by a mutant silkworm, Nd(2). J. Cell. Biol. 99, 2005–2010. Takei, F., Kikuchi, Y., Kikuchi, A., Mizuno, S., Shimura, K., 1987. Further evidence for importance of the subunit combination of silk fibroin in its efficient secretion from the posterior silk gland cells. J. Cell. Biol. 105, 175–180. Tamura, T., Thibert, C., Royer, C., Kanda, T., Abraham, E., Kamba, M., Koˆmoto, N., Thomas, J.-L., Mauchamp, B., Chavancy, G., Shirk, P., Fraser, M., Prudhomme, J.-C., Couble, P., 2000. A piggyBac-derived vector efficiently promotes germ-line transformation in the silkworm Bombyx mori L. Nat. Biotechnol. 18, 81–84. Tanaka, K., Inoue, S., Mizuno, S., 1999a. Hydrophobic interaction of P25, containing Asn-linked oligosaccharide chains, with the H–L complex of silk fibroin produced by Bombyx mori. Insect Biochem. Mol. Biol. 29, 269–276. Tanaka, K., Kajiyama, N., Ishikura, K., Waga, S., Kikuchi, A., Ohtomo, K., Takagi, T., Mizuno, S., 1999b. Determination of the site of disulfide linkage between heavy and light chains of silk fibroin produced by Bombyx mori. Biochim. Biophys. Acta 1432, 92–103. Thomas, J.L., Da Rocha, M., Besse, A., Mauchamp, B., Chavancy, G., 2002. 3 P3-EGFP marker facilitates screening for transgenic silkworm Bombyx mori L. from the embryonic stage onwards. Insect Biochem. Mol. Biol. 32, 247–253. Tomita, M., Munetsuna, H., Sato, T., Adachi, T., Hino, R., Hayashi, M., Shimizu, K., Nakamura, N., Tamura, T., Yoshizato, K., 2003. Transgenic silkworms produce recombinant human type III procollagen in cocoons. Nat. Biotechnol. 21, 52–56. Yamaguchi, K., Kikuchi, Y., Takagi, T., Kikuchi, A., Oyama, F., Shimura, K., Mizuno, S., 1989. Primary structure of the silk fibroin light chain determined by cDNA sequencing and peptide analysis. J. Mol. Biol. 210, 127–139.
A fibroin secretion-deficient silkworm mutant, Nd-sD, provides an efficient system for producing recombinant proteins Satoshi Inouea,1, Toshio Kandaa, Morikazu Imamuraa,2, Guo-Xing Quana,3, Katsura Kojimaa, Hiromitsu Tanakaa, Masahiro Tomitab, Rika Hinob, Katsutoshi Yoshizatob, Shigeki Mizunoc, Toshiki Tamuraa, a Insect Biotechnology and Sericology Department, National Institute of Agrobiological Sciences, 1-2 Owashi, Tsukuba, Ibaraki 305-8634, Japan Hiroshima Tissue Regeneration Project, Hiroshima Prefecture Collaboration of Regional Entities for Advancement of Technological Excellence, Japan Science and Technology Corporation, 3-10-32 Kagamiyama, Higashihiroshima, Hiroshima 739-0046, Japan c Department of Agricultural and Biological Chemistry, College of Bioresource Sciences, Nihon University, 1866 Kameino, Fujisawa 252-8510, Japan
b
Received 3 September 2004; received in revised form 12 October 2004; accepted 15 October 2004
Abstract The silkworm Nd-sD mutant is silk fibroin-secretion deficient. In the mutant, a disulfide linkage between the heavy (H) and light (L) chains, which is essential for the intracellular transport and secretion of fibroin, is not formed because of a partial deletion of the L-chain gene. To utilize the inactivity of the mutant L-chain, we investigated the possibility of using the Nd-sD mutant for the efficient production of recombinant proteins in the silkworm. A germ line transformation of the mutant with a normal L-chain-GFP fusion gene was performed. In the transgenic mutant, normal development of the posterior silk gland (PSG) was restored and it formed a normal cocoon. The biochemical analysis showed that the transgenic silkworms expressed the introduced gene in PSG cells, produced a large amount of the recombinant protein, secreted it into the PSG lumen, and used it to construct the cocoon. The molar ratio of silk proteins, H-chain:L-chain-GFP:fibrohexamerin, in the lumen and cocoon in the transgenic silkworm was 6:6:1, and the final product of the fusion gene formed about 10% of the cocoon silk. This indicates that the transgenic mutant silkworm possesses the capacity to produce and secrete the recombinant proteins in a molar ratio equal to that of the fibroin H-chain, contributing around half molecules of the total PSG silk proteins. r 2004 Elsevier Ltd. All rights reserved. Keywords: Intracellular transport; Transgenic; Recombinant protein; Bioreactor; PiggyBac; Fibroin; Silkworm; Bombyx; Nd-s mutant
1. Introduction The use of a transgenic silkworm as a bioreactor for the production of recombinant proteins has been realized with the success of a germ line transformation Corresponding author. Tel./fax: +81 298 38 6091.
E-mail address: [email protected] (T. Tamura). Present address: MRC Toxicology Unit, University of Leicester, Leicester LE1 9HN, UK. 2 Present address: Prion Disease Research Center, National Institute of Animal Health, Tsukuba, Ibaraki 305-0856, Japan. 3 Present address: Molecular Entomology, Great Lakes Forestry Centre, Canadian Forest Service, Marie, Ontario, Canada P6A 5M7. 1
0965-1748/$ - see front matter r 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.ibmb.2004.10.002
method using transposon piggyBac (Tamura et al., 2000). The domesticated silkworm, Bombyx mori, possesses efficient protein production organs called silk glands, for making silk proteins. In this insect, more than half of the dry weight of the cocoon consists of silk proteins. Therefore, more than half of the nutrients consumed by the larvae are converted into silk proteins. Recently, it was reported that recombinant protein could be produced in the silk gland of transgenic silkworms (Tomita et al., 2003). However, the production was not efficient. The major components of the cocoon of the transgenic silkworm consisted of the products of the native silk genes, suggesting that one of
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the critical factors for the production of recombinant protein is related to the system of silk protein secretion. The major components of cocoon silk in nontransgenic normal silkworms are fibroin and sericin. Fibroin is synthesized in the posterior silk gland (PSG) and accumulates in the lumen of the middle silk gland (MSG), where sericin is synthesized. Then, the fibroin is secreted into the cocoon via the anterior silk gland (ASG). The silk fibroin contains three polypeptides: a 350-kDa heavy chain (H-chain, Shimura, 1983), a 26kDa light chain (L-chain, Yamaguchi et al., 1989), and fibrohexamerin (fhx), originally named P25(Inoue et al., 2000). In the PSG, these proteins form a large protein complex called an elementary unit of fibroin, which consists of a 6:6:1 molar ratio of the H-chain:Lchain:fibrohexamerin (Inoue et al., 2000). The L-chain is linked with the H-chain by a disulfide bond between Cys-172 of the L-chain and Cys-c20 (twentieth residue from the carboxy terminus) of the H-chain (Tanaka et al., 1999b). One molecule of fibrohexamerin interacts non-covalently with six sets of H-L heterodimers to form the elementary unit in the endoplasmic reticulum (ER) (Inoue et al., 2004), and the three N-linked oligosaccharide chains of fibrohexamerin contribute to maintaining the integrity of the elementary unit (Inoue et al., 2000). This molecular complex is essential for quality control in the ER (Reddy and Corley, 1998; Ellgaard et al., 1999; Fassio and Sitia, 2002), enabling the efficient intracellular transport and secretion of significant amounts of fibroin into the lumen (Inoue et al., 2004). The Nd-sD mutant is characterized by an immature PSG and less than 1% of the normal secretion level of fibroin, which leads to the production of a very thin, naked-pupa cocoon that consists mostly of sericin. The gene is mapped to the L-chain gene (fib-L) locus on chromosome 14 (Takei et al., 1984a). The mutation is caused by a deletion downstream from exon III, which causes the recombination of sequences in the third intron with sequences in the far downstream region. This recombination created a chimeric gene containing the first three exons of fib-L and two new exons, IV0 and V0 , from the far downstream region. This mutant chimeric Nd-sD L-chain lacks Cys-172, which was encoded by the original sixth exon, and consequently cannot form a disulfide linkage with the H-chain (Mori et al., 1995). The H–L disulfide linkage contributes to the efficient intracellular transport and secretion of silk fibroin, which is analogous to IgM production (Sitia et al., 1990), and the accumulation of L-chain-deficient fibroin in the ER might inhibit the development of PSG cells. It has been reported that the normal L-chain introduced into the Nd-sD mutant is secreted in the lumen and this suggests that the mutant might be used to increase the production and secretion of L-chainfused recombinant protein.
In this study, the L-chain-GFP fusion gene was introduced into the Nd-sD mutant to assess its ability to produce recombinant protein. The L-chain– GFP fusion gene was expressed in the PSG of the mutant transgenic silkworm; large amounts of recombinant protein were secreted into the lumen and spun into the cocoon. The Nd-sD phenotype, which consisted of an immature PSG and thin cocoon, was rescued dramatically and the cocoon was almost indistinguishable from those of normal silkworms. A single cocoon contained about 8.6 mg dry weight (16.6 mmol) of L-chain-GFP fusion protein, which corresponds to about 10% of the total cocoon silk protein. The molar ratio of H-chain:Lchain-GFP:fibrohexamerin in the secreted fibroin was strictly 6:6:1, indicating that the formation of complexes of the three proteins is critical, even with the fusion protein.
2. Materials and methods 2.1. Silkworms Fertilized eggs of fibroin-secreting wild-type B. mori C108 and the fibroin-secretion-deficient, naked-pupa mutant of B. mori Nd-sD (with a mutation of the fibroin L-chain gene, fib-L) (Takei et al., 1984a; Mori et al., 1995) were supplied by the Insect Genetics Laboratory of the National Institute of Agrobiological Sciences, Japan. The larvae were reared on an artificial diet (Nihon Nosan) at 25 1C. 2.2. Plasmid vectors The helper plasmid vector, pHA3PIG (6.2 kb), is described elsewhere (Tamura et al., 2000). The DsRed2 cDNA that was excised from the pDsRed2-1 vector (Clontech) was used to replace EGFP cDNA in pBac(3 P3-EGFPafm) (Horn and Wimmer, 2000), giving rise to pBac(3 P3-DsRed2). The L-chain 50 flanking region (from 600 to +34) (Kikuchi et al., 1992), the L-chain cDNA sequence (from +34 to +767) [2], and the L-chain 30 -flanking region (from +13114 to +13597) (Kikuchi et al., 1992) were amplified by PCR from genomic DNA prepared from B. mori. EGFP cDNA was excised from the pEGFP vector (Clontech). All of the cDNA fragments were inserted into pBac(3xP3-DsRed2) to produce pBac(3xP3DsRed2+L-chain-GFP) (Fig. 1A). 2.3. Transformation Plasmid DNA was purified using the QIAGEN Midi (Qiagen). To break embryonic diapause, eggs were treated within 4 h after oviposition with 20% HCl for 1 h at 25 1C. A mixture of helper plasmid pHA3PIG
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2.5. Northern blot analysis Total RNA from PSG or MSG at the 3rd day of the 5th instar was purified using ISOGEN (Nippongene). Total RNA was subjected to Northern blotting and hybridized with the probe as follows. Part of the L-chain cDNA sequence (+1 to +357; corresponding to exons I to III) that is common to both the normal and Nd-sD Lchains was amplified from pFL18 by PCR (Yamaguchi et al., 1989). Part of the GFP cDNA sequence (+1 to +357) was amplified from pEGFP (Clontech) by PCR. Part of the B. mori elongation factor a-I isoform cDNA sequence (from +1 to +1682) was amplified from pBmEF-1 a-1 by PCR (Kamiie et al., 1993). Fig. 1. Restriction enzyme map of a piggyBac vector and genomic DNA of the normal breed (C108) and Nd-sD. (A) Design of a piggyBac vector for L-chain-GFP expression in PSG cells. The DsRed2 marker gene construct is placed immediately downstream from the Pax-6 artificial promoter sequences (3 P3), enabling its expression in photoreceptor cells. The L-chain promoter is fused to the L-chainGFP coding sequence and L-chain 30 -untranslated sequences. Inserted terminal repeats of piggyBac transposase (arrows) and EcoRI sites (E) are indicated. (B) Physical maps of the normal (C108) and Nd-sD mutant L-chain genes. The normal gene (Fib-L) contains seven exons (exon I-VII), whereas the chimeric mutant gene contains exons I–III of Fib-L and exons IV0 and V0 derived from the far downstream region.
2.6. Protein analysis Preparation of fibroin samples from PSG lumen, PSG tissues, and cocoons has been described elsewhere (Tanaka et al., 1999a; Inoue et al., 2000). Western blotting of fibroin samples was carried out, as described previously (Inoue et al., 2000), with the following antibodies: rabbit anti-H-chain or anti-L-chain polyclonal antibody (Inoue et al., 2000), mouse anti-fhx polyclonal antibody (Inoue et al., 2000), or rabbit antiGFP (Invitrogen) antibodies. 2.7. Purification of L-chain-GFP
(2–3 nl) [11] and vector DNA (0.2 mg/ml of each) in 0.5 mM phosphate buffer (pH 7.0) with 5 mM KCl was injected into 476 eggs 4 h after the acid treatment. Injected eggs were placed in a humidified box at 25 1C until hatching. Hatched larvae were transferred to an artificial diet and grown to adulthood. G0 moths were mated within the same family, and 7-day G1 embryos were screened for DsRed2 expression in the stemmata and nervous system tissues (Thomas et al., 2002) with a Leika MZFL III fluorescence microscope (Leika) equipped with filter sets for DsRed. DsRed2 expression in screened silkworms was confirmed in the compound eyes of G1 moths. Moths expressing DsRed2 were mated with each other. The G2 larvae that were most strongly positive for DsRed2 were used as transgenic lines. Green fluorescent protein in the dissected silk gland and cocoon was observed with a Leika MZFL III fluorescence microscope (Leika) equipped with filter sets for GFP2.
To purify the L-chain-GFP fusion protein, a fibroin sample from the PSG lumen was subjected to 0.1% SDS, 12.5% PAGE (SDS-PAGE); the L-chain-GFP fusion protein was electro-eluted in 5 mM Tris, 38.4 mM glycine. The purity of the L-chain-GFP fusion protein was judged by silver staining and Western blotting. 2.8. Quantitative ELISA Standard curves for the H-chain, L-chain, and fibrohexamerin are described elsewhere (Inoue et al., 2000). Standard curves for L-chain-GFP were made using the purified L-chain-GFP protein; the protein was dissolved in 20 mM Tris–HCl (pH 8.0), 2 M urea, and subjected to ELISA using rabbit anti-L-chain antibody. Protein samples prepared from the PSG lumen or the cocoon shell were dissolved in 20 mM Tris–HCl (pH 8.0), 2 M urea, and subjected to quantitative ELISA as described elsewhere [3].
2.4. Southern blot analysis Genomic DNA was extracted from G2 moths (Tamura et al., 2000). EcoRI-digested genomic DNA (5 mg) was subjected to Southern blotting and hybridized with the L-chain 30 –flanking region (from +887 to +1354) from pFL18 (Yamaguchi et al., 1989) as a probe for the normal L-chain gene.
3. Results 3.1. Construction of Nd-sD mutant transgenic silkworms We constructed the transformation vector pBac(3 P3-DsRed2+L-chain-GFP) (Fig. 1A) and
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injected it with helper plasmid into 476 eggs of Nd-sD at the preblastodermal stage. Fifteen transgenic silkworms were verified by screening for DsRed2 emission in the stemmata and nervous tissues of embryos or moths (Fig. 2B), as reported by Thomas et al. (2002) in the transgenic line containing the 3XP3GFP insert, and even in a silkworm with normal brown color stemmata
and compound eyes. These silkworms were mated with each other to produce the LGL4 5 line, which was characterized by a strong DsRed2 emission in the stemma and complex eyes. To confirm the insertion of the fusion gene and to identify the number of integrated DNA molecules in the Nd-sD genome of the transgenic line, the EcoRI-digested genomic DNA of wild type, NdsD, and the LGL4 5 line were investigated by Southern blot hybridization with a normal L-chain-specific probe (Fig. 1B). The probe hybridized with one specific 8.7-kb band in wild-type silkworms (Fig. 2B lane 1), no band in Nd-sD mutants (Fig. 2B lane 2), as expected, and 9.8-, 7.8-, 5.6-, and 4.1-kb bands in the LGL4 5 line (Fig. 2B lane 3). This indicated that the LGL4 5 line was a transgenic line containing the normal L-chain-GFP fusion gene in at least four different loci of the original Nd-sD genome. 3.2. Restoration of PSG development and fibroin secretion in the transgenic line The most prominent phenotypic features of the Nd-sD mutant silkworm are an immature PSG (Fig. 3A(b)) and the production of a very thin cocoon (Fig. 3B(b)) relative to that of a normal silkworm (Figs. 3A(a), B(a)). The phenotypes of the transgenic line LGL4 5 were dramatically changed from those of Nd-sD with respect to the extensive development of the PSG (Fig. 3A(c)) and the production of a thick cocoon (Fig. 3B(c)) similar to that of the normal C108 (Figs. 3A(a), B(a)). The mean weight of cocoons produced by transgenic line LGL4 5 was 78.4 mg (n ¼ 50), which was much heavier than that of Nd-sD (17.7 mg; n ¼ 50), but less than half that of the normal C108 (187 mg; n ¼ 50). These results indicate that the prominent phenotypes of the Nd-sD mutant can be largely converted to those of the normal fibroin-producing silkworm by transgenesis with the normal L-chain-GFP fusion gene. 3.3. Expression and secretion of functional L-chain– GFP fusion protein in transgenic silkworms
Fig. 2. Morphological characters of transgenic Nd-sD mutant inserted using the 3XP3DsRed2 marker gene with genomic Southern blotting. (A) Expression of DsRed2 in the compound eyes of G1 moths (upper) and in the stemmata of a 7-day-old G2 embryo (lower). Note that the mutant possesses black stemmata and compound eyes. The marker is still useful for detecting transgenic silkworm. Left panel; Nd-sD, right panel; transgenic line. An arrowhead and arrows point to the ocelli and abdominal nervous system, respectively. Scale bars: 0.5 mm. (B) Southern blot of EcoRI-digested genomic DNA (5 mg each) from C108 (lane 1), Nd-sD (lane 2), and the transgenic line (lane 3) hybridized with the normal L-chain specific probe, as shown in Fig. 1B.
The emission of green fluorescence due to the expression of the integrated GFP gene was observed in the transgenic silkworm at the 5th day of the 5th instar, when the secretion of fibroin reached the maximal level, in the PSG cells and the lumen of whole silk glands (from PSG, MSG to ASG) (Fig. 3A(c) and (c*)). The fluororescence was not detected in other tissues at whole stages, suggesting that the fusion gene specifically expressed in PSG cells. Green fluorescence was also detected in the entire cocoon produced by the transgenic silkworm (Fig. 3B(c)). These observations suggested that the L-chain-GFP fusion protein was expressed from the integrated fusion gene in the transgenic silkworm.
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Fig. 3. GFP fluorescence in the PSG and cocoon of the transgenic line. (A) A pair of silk glands at the 5th day of the 5th instar larvae of C108 (a), Nd-sD (b), and the transgenic line (c) were observed under bright field (bright) and with the GFP fluorescence system (GFP). The ASG, MSG, and PSG of the transgenic line were observed under higher magnification with the EGFP fluorescence system (c*). ASG: anterior silk glands, MSG: middle silk glands, PSG; posterior silk glands. (B) Cocoons produced by C108 (a), Nd-sD (b), and the transgenic line (c) were observed similarly.
In order to prove this, Northern blots of total RNA preparations from PSG or MSG cells of the normal breed C108, Nd-sD, or the transgenic line LGL4 5 were probed with cDNA for the L-chain sequence derived from exons I, II, and III (common to C108 and Nd-sD Lchains) or cDNA for GFP. The L-chain cDNA probe hybridized to 1.3- and 1.1-kb bands in the RNA preparations from the PSGs of C108 and Nd-sD, respectively (Fig. 4A, left panel, lanes 1 and 2). The amount of Nd-sD L-chain mRNA was less than 5% of that in C108. The L-chain cDNA probe did not hybridize to RNA preparations from the MSG of either C108 or Nd-sD (Fig. 4A, left panel, lanes 4 and 5). These results are consistent with those reported previously (Takei et al., 1987).
Fig. 4. Detection of L-chain-GFP transcript and protein in the transgenic line. (A) Northern blots of total RNA (5 mg/lane) from the PSG (lanes 1–3) and MSG (lanes 4–6) of C108 (lanes 1 and 4), Nd-sD (lanes 2 and 5), and the transgenic line (lanes 3 and 6) hybridized with the L-chain cDNA probe (exons I–III, 357 bp) common to both normal and Nd-sD L-chain mRNA, GFP cDNA probe (357 bp), or the cDNA probe for the B. mori elongation factor a-I isoform. (B) Western blot of cocoon proteins of C108 (a), Nd-sD (b), and the transgenic line (c). The PSG tissue (lanes 1 and 3) and the cocoon (lanes 2 and 4) proteins were separated by SDS-PAGE without reduction (lanes 1 and 2) or under reducing conditions (lanes 3 and 4) and subjected to Western blotting with anti-H-chain (left), anti-L-chain (middle), or anti-GFP antibodies (right).
The L-chain cDNA probe hybridized to 2.1- and 1.1kb components in the RNA preparation from the PSG of transgenic line LGL4 5 (Fig. 4A, left panel, lane 3).
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The 2.1-kb, but not the 1.3-kb, component hybridized with the GFP cDNA probe (Fig. 4A, right panel, lane 3). The hybridizing RNA bands were interpreted as normal L-chain mRNA (1.3 kb), Nd-sD L-chain mRNA (1.1 kb), and normal L-chain-GFP fusion gene mRNA (2.1 kb). Judging from the relatively strong intensity of the 2.1-kb band, it appeared that the normal L-chainGFP fusion transgenes were actively transcribed in a PSG-specific manner in the transgenic line LGL4 5. To detect L-chain or L-chain-GFP fusion polypeptides, proteins from PSG tissue or from cocoon shells of C108, Nd-sD, or LGL4 5 were subjected to Western blotting using anti-H-chain, anti-L-chain, or anti-GFP antibodies, before and after the reductive cleavage of disulfide bonds. In the normal C108 strain, the L-chain was detected in two forms, either unbound or bound to H-chain, in the PSG tissue sample (Fig. 4B(a), lanes 1 and 3), but only the H-chain-bound form was detected in the cocoon sample (Fig. 4B(a), lanes 2 and 4), as reported (Takei et al., 1987). In Nd-sD, the mutant Lchain was detected in an unbound form in the PSG tissue sample (Fig. 4B(b), lanes 1 and 3), but not in the cocoon sample (Fig. 4B(b), lanes 2 and 4), as reported (Takei et al., 1987). In the transgenic line, the anti-Lchain antibody reacted with 52.5- and 27-kDa bands in the PSG tissue sample after reducing the disulfide bonds (Fig. 4B(c), middle panel, lane 3), whereas the anti-GFP antibody reacted with only the 52.5-kDa band under the same conditions (Fig. 4B(c), right panel, lane 3). These results strongly suggest that the 52.5-kDa band is the normal L-chain-GFP fusion protein and that the 27kDa band is the Nd-sD mutant L-chain. As expected, only the normal L-chain-GFP fusion protein was detected in the cocoon protein sample in the reactions with anti-L-chain antibody or anti-GFP antibody (Fig. 4B(c), middle and right panels, lane 4). The normal Lchain-GFP fusion protein was associated with the Hchain before reduction of the disulfide bonds, as shown by the reaction with both anti-L-chain and anti-GFP antibodies (Fig. 4, middle and right panels, lane 2). These results suggest that the normal L-chain-GFP fusion genes, which were integrated into the genome of the transgenic silkworm, are expressed as proteins that behave like the normal L-chain and contribute to the restoration of the phenotype with respect to high secretion levels of fibroin and the production of nearly normal cocoons. 3.4. Quantification of the normal L-chain-GFP fusion protein in cocoons The molar ratio of H-chain:L-chain:fibrohexamerin in the elementary unit of fibroin produced by the normallevel fibroin-producing breed of silkworms is 6:6:1 (Inoue et al., 2000). We used quantitative ELISA to determine whether the normal L-chain-GFP fusion
protein expressed in the transgenic silkworm participated in the formation of an elementary unit with a similar molar ratio. For this purpose, the normal L-chain-GFP fusion protein was purified from the fibroin secreted into the PSG lumen of the transgenic silkworm, as described in the Methods and materials. The purity of the protein sample was examined by SDS-PAGE, followed by silver staining or Western blotting. It behaved as a single 52.5kDa band (Fig. 5A(a)) and reacted with the anti-L-chain or anti-GFP antibody, but not with the anti-H-chain or anti-fibrohexamerin antibody (Fig. 5A(b)–(e)). This protein sample was then used to make an ELISA standard curve for the L-chain-GFP fusion protein (Fig. 5B). ELISA standard curves for H-chain and fibrohexamerin were made, as described (Inoue et al., 2000). The results of the quantitative ELISA for cocoon protein samples are summarized in Table 1. The molar ratio of H-chain:L-chain:fibrohexamerin was close to 6:6:1 for the normal breed C108, 6:0:1 for Nd-sD, and close to 6:6:1 for the transgenic line LGL4 5. These results
Fig. 5. Purification of L-chain-GFP and the production of a standard curve for quantitative ELISA. (A) Purification of L-chain-GFP. A fibroin sample (lane 1) from the transgenic line was separated by SDSPAGE without cleaving the disulfide bonds, and L-chain-GFP (lane 2) was recovered by electro-elution. The purity was judged by silver staining (a), and Western blotting using anti-H-chain (b), anti-L-chain (c), anti-fibrohexamerin (d), and anti-GFP (e) antibodies. (B) ELISA for the purified L-chain-GFP. The purified L-chain-GFP was subjected to ELISA using anti-L-chain antibody. The reactivity was monitored by the reaction with horseradish peroxidase-conjugated antirabbit IgG followed by the color-developing reaction (A490) with o-phenylenediamine dihydrochloride.
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2.23 104 ND 2.14 104 2.21 104 70 2.17 104 5.75 103 ND 1.1 104 C108 Nd-sD Transgenic line
0.517 0.401 0.622
0.354 ND 0.274
0.196 0.421 0.18
6.31 104. 245 7.58 104
1.12 103 3 1.04 103
H-chain L-chain or L-chain-GFP H-chain L-chain or L-chain-GFP
Fhx H-chain
fhx
pmol ng/protein (100 mg) A490 a
Breed
Table 1 Molar ratios of H-chain, L-chain or L-chain-GFP, and fhx in the fibroin of cocoons
We demonstrated that the Nd-sD mutant, which possesses degenerated PSG and expels only sericin in the cocoon, is useful for producing a large amount of Lchain fused recombinant proteins in the transgenic silkworm. The amount of the L-chain-GFP fusion proteins in our experiment exceeded 10% of the proteins in the cocoon when the gene was introduced into the mutant. In addition, the Nd-sD mutant was rescued by the introduction of L-chain-GFP fusion protein gene, suggesting that the protein production and secretion machinery of the mutant PSG had recovered. We used the fusion gene with a promoter from the 600-bp upstream region of the L-chain gene. The expression of the inserted fusion gene was apparently lower than that in the normal type assessed by Northern blotting (Fig. 4A compare lanes 1 and 3). However, the tissue-specificity of the inserted gene was maintained in the transgenic silkworm, indicating that signals related to the tissue-specific expression of the L-chain gene are localized to this region. There are two possible explanations for the low level of gene expression: (i) the inserted gene disturbed a critical chromatin structure at the insertion position, which affected its expression, or (ii) the binding sites of some transcriptional activators were not present in the promoter region used. For the latter possibility, using a longer upstream region as the promoter may increase the mRNA level. The fibroin H- and L-chain genes and the fibrohexamerin gene share the same transcriptional regulation system, and are expressed in a coordinated manner in the PSG (Bello et al., 1994), despite the fact that the three genes are located on different chromosomes: FibH is on chromosome 25, fib-L is on chromosome 14, and fibrohexamerin is on chromosome 2. Otherwise, it might be better to use the promoter of H-chain, or fibrohexamerin genes for the L-chain, or the targeted gene expression system of GAL4/UAS (Imamura et al., 2003). Reduced expression of the introduced gene, as compared to the endogenous gene, is also observed when the promoters of other genes are used (Suzuki et al., 2003). Another important factor is the secretion of the recombinant proteins in PSG. The ER provides an
L-chain or L-chain-GFP
4. Discussion
57 n ¼ 6 times; 7, SD; ND, not detected. a Values obtains by ELISA. Samples (all in 100 ml) assayed were 10 ng (for H-chain), 50 ng (for L-chain) and125 ng (for fhx) proteins from the cocoons of C108 and the transgenic line; 2 mg (for Hchain), 10 mg for L-chain, 100 mg for fhx proteins from Nd-sD.
5.9270.04:5.9870.05:1 6.3170.07:—:1 6.2070.07:6.1770.03:1 3.73 103 11.1 3.47 103
fhx
Molar ratio
suggest that the L-chain-GFP fusion proteins expressed in the transgenic silkworm participated in the assembly of an elementary unit of fibroin whose molecular constitution was similar to that formed in the normal breed, irrespective of the presence of Nd-sD mutant Lchain proteins in the PSG cells. The content of L-chainGFP fusion protein was calculated to be about 110 mg (212 nmol)/mg of the cocoon protein or about 8.6 mg (16.6 mmol) per dried cocoon (78.4 mg) produced by the transgenic silkworm.
H-chain : L-chain or L-chain-GFP : fhx
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environment and mechanisms to facilitate the proper formation of newly synthesized proteins into higher order conformations. The quality control function of the ER (Hammond and Helenius, 1995) is critical to ensuring that only properly folded proteins enter secretory pathways. In the case of silk fibroin, a disulfide linkage between the H- and L-chains (Takei et al., 1984b, 1987), the addition of three N-linked oligosaccharide chains onto fibrohexamerin (Tanaka et al., 1999a; Inoue et al., 2000), and the assembly of the elementary unit (Inoue et al., 2000) all take place in the ER (Inoue et al., 2004). These processes are important for the efficient secretion of fibroin. The significance of the H–L linkage for the efficient secretion of fibroin has been suggested by genetic and biochemical studies of the fibroin secretion deficient Nd-sD mutant (Takei et al., 1987; Mori et al., 1995; Tanaka et al., 1999b). The application of the transgenic silkworm as a bioreactor to produce recombinant proteins has several advantages: (i) the cost of silkworm rearing is much lower than the cost of rearing other animals; (ii) large quantities of recombinant protein can be produced (0.5–2 mmol fibroin per dried 200–500 mg cocoon); (iii) post-translational modifications are similar to those of mammalian proteins, except for oligosaccharide processing; (iv) the protein composition is relatively simple; and (v) the risk of disease is lower. Recently, Tomita et al. (2003) constructed a wild-type transgenic silkworm containing the L-chain-GFP and Lchain-human type III procollagen genes; the exogenously expressed L-chain-GFP protein was secreted and was found in the dried cocoon. However, the secretion efficiency of the fusion proteins in the wild-type transgenic silkworm was very low. By contrast, the Nd-sD mutant transgenic silkworm we established showed much higher production of the recombinant protein. The reason for this higher production by the mutant is outlined in Fig. 6. The fusion protein produced in the normal breed competes with the normal L-chain in the process of formation of the H–L linkage. The endogenous normal L-chain has a greater affinity for the H-chain than does the L-chain-GFP fusion protein. Therefore, the wild-type transgenic silkworm secreted relatively more endogenous normal L-chain than the fusion protein. Actually, the molar ratio of endogenous L-chain to L-chain-GFP fusion protein in the cocoon of the transgenic normal breed is about 10:1, concomitant with the accumulation of a relatively large amount of fusion protein in PSG cells (S. Inoue et al., unpublished data). However, the mutant L-chain in the transgenic Nd-sD mutant cannot compete with the fusion protein. Therefore, only the fusion protein is secreted into the lumen. Indeed, the fusion protein was secreted efficiently. In this study, a cocoon of the mutant contained about 8.6 mg (16.6 mmol) of the functional (green fluorescence emitting) L-chain-GFP fusion pro-
Fig. 6. Model of the intracellular transport of recombinant proteins produced in the transgenic silkworms. H, fibroin H-chain; L, fibroin Lchain; Lm, fibroin L-chain produced by Nd-sD mutant; fhx, fibrohexamerin. The L-chain-GFP fusion protein (L+GFP) produced in the PSG cells of the normal breed has lower affinity in the formation of an S–S linkage with the H-chain, compared with the normal Lchain. Therefore, less of the fusion protein is secreted in the normal breed. However, the fusion protein in the mutant does not compete with the mutant L-chain. Therefore, only the fusion protein linked with H-chain is secreted in the mutant.
tein (Table 1). The efficient secretion of L-chain-GFP fusion protein provides the Nd-sD mutant transgenic line with the advantage of higher protein yields than in the wild-type transgenic silkworm. In addition, the L-chainGFP fusion protein is more easily purified from cocoons of the Nd-sD mutant transgenic line, because the cocoons lack the endogenous L-chain. Our future work will focus on designing other functional proteins and establishing an easy purification system with high yields of the recombinant proteins from cocoons. The tandem affinity purification (TAP) system may prove to be a powerful tool for the purification of functional foreign recombinant proteins from cocoons (Rigaut et al., 1999).
Acknowledgments We would like to thank Dr. Ernst A. Wimmer of Universitat Bayeyth for kindly providing pBac(3 P3EGFPafm), Kazuko Seo and Hiroko Yamazaki for technical assistance, and Dr. Michelle A. Hughes (University of Leicester) for a critical reading of the manuscript. This work was supported by the Ministry of Agriculture, Forest, and Fisheries and by the Program for the Promotion of Basic Research Activities for Innovative Bioscience, Japan.
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