Production of Bombyx mori Silk Fibroin Incorporated with Unnatural

Jun 2, 2014 - Silk fibroin incorporated with unnatural amino acids was produced by in vivo .... Incorporation of non-canonical amino acids into protei...
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Production of Bombyx mori Silk Fibroin Incorporated with Unnatural Amino Acids Hidetoshi Teramoto* and Katsura Kojima Silk Materials Research Unit, Genetically Modified Organisms Research Center, National Institute of Agrobiological Sciences (NIAS), Tsukuba, Ibaraki 305-8634, Japan S Supporting Information *

ABSTRACT: Silk fibroin incorporated with unnatural amino acids was produced by in vivo feeding of p-chloro-, p-bromo-, and p-azido-substituted analogues of L-phenylalanine (Phe) to transgenic silkworms (Bombyx mori) that expressed a mutant of phenylalanyl-tRNA synthetase with expanded substrate recognition capabilities in silk glands. Cutting down the content of Phe in the diet was effective for increasing the incorporation of Phe analogues but simultaneously caused a decrease of fibroin production. The azide groups incorporated in fibroin were active as chemical handles for click chemistry in both the solubilized and the solid (fibrous) states. The azides survived degumming in the boiling alkaline solution that is required for complete removal of the sericin layer, demonstrating that AzPhe-incorporated silk fibroin could be a versatile platform to produce “clickable” silk materials in various forms. This study indicates the huge potential of UAA mutagenesis as a novel methodology to alter the characteristics of B. mori silk.

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INTRODUCTION The domesticated silkworm, Bombyx mori, is an important organism from an industrial point of view because of its remarkable ability to synthesize proteins: one larva produces hundreds of milligrams of silk proteins to make a robust cocoon.1 Due to its mechanical strength and biocompatibility, B. mori silk has been used as a surgical suture for decades.2 Recent progress in the processing of silk into various forms3 such as films,4,5 hydrogels,6,7 regenerated fiber,8 nanofiber,9 tubes,10 and three-dimensional scaffolds11,12 has triggered extensive studies of silk-based materials for biomedical applications.13 Moreover, recent success in the germline transgenesis of B. mori14 has enabled genetic engineering of silk, such as modifications to enhance its mechanical properties15,16 or to add novel functions.5,17−19 The transgenic techniques have also been utilized for massive production of recombinant proteins in silk glands.20,21 Since silk proteins carry reactive functional groups as side chains of amino acids, chemical modification is another powerful technique to alter their characteristics.22 In this study, we investigated the possibility of introducing unnatural amino acid (UAA) mutagenesis methodology as an alternative tool to modify B. mori silk. Over the past decade and a half, UAA mutagenesis of proteins has been developed as a powerful methodology for probing protein functions and creating novel proteins. In vivo incorporation of UAAs into proteins has been achieved using two major approaches, that is, a site-specific23 and a residuespecific method.24 The site-specific method employs orthogonal tRNA/aminoacyl-tRNA synthetase (aaRS) pairs specific for © 2014 American Chemical Society

target UAAs, which are incorporated into proteins at specific sites in response to unique codons. In contrast, the residuespecific method is relatively simple: it requires only that aaRSs be engineered to expand their recognition capabilities.25,26 With this method, UAAs structurally analogous to targeted amino acids can be incorporated in response to their corresponding natural codons, thereby enabling incorporation of multiple UAAs into proteins. To date, a range of unicellular27−29 and multicellular30−32 organisms have been engineered to incorporate UAAs bearing a wide variety of functional groups into proteins in vivo by using these two methods. We have begun to explore the possibility of applying residuespecific UAA mutagenesis to B. mori, because this technique requires less genetic manipulation of the host organism than the site-specific method. We recently reported the residuespecific incorporation of several unnatural analogues of Lphenylalanine (Phe) in protein biosynthesis of B. mori cultured cells, in which engineered B. mori phenylalanyl-tRNA synthetase (BmPheRS) mutants with expanded substrate recognition capabilities were expressed.33,34 We have chosen Phe as a target amino acid to be replaced with its unnatural analogues for the following reasons: (1) Phe is an essential amino acid of B. mori,35 which makes it possible to control its uptake by feeding; (2) a wide range of knowledge around the structure−function relationships of PheRS has been accumulated;36,37 and (3) a variety of Phe analogues are commercially Received: April 11, 2014 Revised: May 20, 2014 Published: June 2, 2014 2682

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AzPhe was added, the diet was kept away from light exposure as much as possible to avoid photolysis of AzPhe. When the average body weights of larvae stopped increasing, each larva was individually transferred into a handmade paper box to allow it to start cocooning (at the seventh day in most cases). After 3 days, the cocoons were harvested, weighed, and stored at −20 °C until analysis. Measurement of Fibroin Amount in Cocoons by Urea Degumming. Small pieces of each individual harvested cocoon were degummed in 100 v/w of 8 M urea in 40 mM Tris-SO4 (pH 7) at 80 °C for 10 min to remove the sericin layer.40 The urea-degummed cocoon pieces were thoroughly washed with deionized water, dried for 1−2 h at 50 °C in vacuo, and weighed. The weight loss by urea degumming was calculated using the following equation:

available. In addition, it was expected that the replacement of Phe with its analogues would not have critical effects on the fiber-forming property of silk because Phe only exists in the noncrystalline regions of silk fibroin molecules.38 Three kinds of BmPheRS mutants were designed based on the mutations introduced to the Escherichia coli PheRS for residue-specific incorporation of Phe analogues in bacteria,26 and one of the mutants, an Ala450 to Gly mutant in the α-subunit of BmPheRS, was found to be a suitable candidate for transplantation to B. mori larvae.34 Here we report, for the first time, the in vivo production of silk fibers containing unnatural Phe analogues, p-chloro-Lphenylalanine (ClPhe), p-bromo-L-phenylalanine (BrPhe), and p-azido-L-phenylalanine (AzPhe; Figure 2a), with a newly generated transgenic B. mori line expressing the A450G mutant of the BmPheRS α-subunit in silk glands. These Phe analogues were incorporated into silk fibroin by simply adding them to the diet. Cutting down the content of Phe in the diet was effective for increased incorporation of the analogues, which in turn resulted in decreased fibroin production. We verified that azide groups incorporated into fibroin can be selectively used for the azide−alkyne cycloaddition reaction (click chemistry) both in solubilized and solid (fibrous) states. Azides can survive the standard alkaline degumming condition necessary for complete removal of the sericin layer, demonstrating that the azide-functionalized silk fibroin can be processed into various silk-based materials via conventional techniques.

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weight loss by urea degumming(%) =

Wb − Wa × 100 Wb

where Wb and Wa denote the weights of the cocoon pieces before and after urea degumming, respectively. The weights of fibroin were calculated from the weights of whole cocoons and the weight loss by urea degumming was obtained as described above. SDS-PAGE. Aliquots of whole (undegummed) cocoon were dissolved in 8 M lithium bromide (LiBr) aqueous solution at 35 °C at a concentration of 50 μg/μL. The solutions were diluted to 0.75 μg/ μL with 8 M urea, then mixed with a 1/2 volume of 6 × SDS-PAGE sample buffer containing reducing agent (Nacalai Tesque), and incubated over 30 min at room temperature. A total of 4 μL of each sample solution was applied to an Any kD Mini-PROTEAN TGX Gel (Bio-Rad, Hercules, CA), then separated at a constant voltage of 200 V, and the protein bands were visualized with EzStain Aqua (ATTO, Tokyo, Japan). In-Gel Digestion and Mass Analysis of FibL. Samples were prepared as above except that the protein concentration was made 6fold higher. A total of 7−14 μL of the sample solution was applied to 12.5% e-PAGEL precast polyacrylamide gels (ATTO), then separated at a constant current of 20 mA, and the protein bands were visualized with EzStain Aqua (ATTO). The stained bands assigned to FibL were excised from the gels and subjected to in-gel digestion with trypsin, followed by MALDI-TOF-MS analysis, as described previously.34 MALDI-TOF-MS spectra were collected in the positive ion reflector mode with an autoflex III mass spectrometer (Bruker Daltonics, Billerica, MA, U.S.A.) in the range of 500 to 5000 Da. The mass spectrometric data were analyzed by mMass software.41 Modification with Alkyne-PEG4-Biotin. Aliquots of ureadegummed cocoon were dissolved in 8 M LiBr aqueous solution at 35 °C at a concentration of 25 μg/μL, and 5 μL of each of the solutions was mixed with 44 μL of the reaction premix (25 μL 8 M urea, 5 μL 1 M Tris-HCl (pH 8), 1 μL 50 mM CuSO4, 1.25 μL 40 mM TBTA in DMSO/t-BuOH (1:4), 1 μL 5 mM alkyne-PEG4-biotin in DMSO, and 10.8 μL deionized water) in 0.5 mL centrifuge tubes. Finally, 1 μL of freshly prepared 50 mM sodium ascorbate solution was added to each reaction tube. The reaction mixtures were incubated overnight at room temperature. Attachment of biotin on FibL, P25, and FibH was verified by Western blotting with HRP-streptavidin, as described in the Supporting Information. Modification with Alkyne-PEG3-Carboxyrhodamine 110. Approximately 1 mg of each piece of urea-degummed cocoon was fixed on a plastic case using double-faced tape. The fixed cocoon pieces were covered with 50 μL of the reaction mixture (0.1 M Tris-HCl (pH 8), 1 mM CuSO4, 1 mM TBTA, 0.1 mM alkyne-PEG3-carboxyrhodamine 110, and 1 mM sodium ascorbate) and incubated for 2 h at room temperature. After the reaction, the cocoon pieces were washed with DMSO followed by deionized water and then dried for 30 min at 50 °C in a vacuum oven. Green fluorescence from the fluorophores attached to silk fibroin was observed with an MZ16 FA fluorescence stereomicroscope (Leica Microsystems) using a GFP2 filter set (480/ 40 nm excitation filter and 510 nm barrier filter). Sodium Carbonate Degumming. Cocoons cut into small pieces were boiled in 0.02 M Na2CO3 for 30 min and washed thoroughly

EXPERIMENTAL SECTION

Materials. All chemicals used in this study were of reagent grade and used as received. Phe was from Nacalai Tesque (Kyoto, Japan). ClPhe and BrPhe were from Bachem AG (Bubendorf, Switzerland). AzPhe was from Bachem AG and Watanabe Chemical Industries (Hiroshima, Japan). Tris[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl]amine (TBTA) was from Sigma-Aldrich (St. Louis, MO). AlkynePEG4-biotin and alkyne-PEG3-carboxyrhodamine 110 were from Click Chemistry Tools (Scottsdale, AZ). Synthetic oligonucleotides were from Hokkaido System Science (Sapporo, Japan) and Sigma-Aldrich. A B. mori strain, MCS601, which has been maintained in the Transgenic Silkworm Research Unit at NIAS, was used for germline transformation. B. mori larvae were reared on an artificial diet (SilkMate PS; Nosan Corporation, Yokohama, Japan) at 22−26 °C. Germline Transformation. The piggyBac plasmid vector for germline transformation of MCS601 was constructed as described in the Supporting Information. Germline transformation of B. mori was performed as described elsewhere39 with minor modifications. The eggs of MCS601 were acid-treated for 30 min at 3 h postoviposition, and then a mixed solution of pBac[3×P3-DsRed2afm]-BmPheRS_α_A450G and a helper plasmid, pHA3PIG, was injected into the eggs at 4−10 h postoviposition. The hatched larvae (G0) were reared and permitted to mate with each other. The resultant embryos (G1) were screened by using an MZ16 FA fluorescence stereomicroscope (Leica Microsystems, Wetzlar, Germany) for transgenic individuals with DsRed2 expression at 6 to 7 days after oviposition. The transgenic individuals were reared together and sib-mated for at least three generations. The established transgenic line was designated as H01. Expression of the transgene was verified by RT-PCR as described in the Supporting Information. Administration of UAAs. UAAs were administered to larvae by mixing them into the synthetic diet as described in the Supporting Information. Male larvae of the H01 and wild-type (WT; MCS601) strains were fed a synthetic diet of standard composition (Table S1) on their first and second days of fifth instar. From the third day, groups of three larvae were transferred to individual plastic cases and fed different diets having specified compositions. Once a day, the average body weights of each group of three larvae were recorded. When 2683

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with deionized water as described elsewhere.3 The degummed cocoons were dried overnight at 25 °C and stored at −20 °C until use.

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RESULTS AND DISCUSSION Generation of a Transgenic Line Expressing the A450G Mutant of the BmPheRS α-Subunit in Posterior Silk Glands. Silk glands of B. mori are functionally divided into three parts, the anterior, middle and posterior silk glands (respectively abbreviated as ASG, MSG, and PSG).42 The major protein component of silk, fibroin, which forms the fibrous core and accounts for about 70 wt % of cocoon proteins, is synthesized in the PSG, whereas sericins, a glue-like protein family accounting for about 30 wt % of cocoon proteins, are synthesized in the MSG. To achieve the incorporation of UAAs into fibroin, a cDNA encoding the BmPheRS α-subunit bearing an A450G mutation was inserted into a conventional piggyBac vector downstream of the FibL promoter for specific expression in PSG.39 The constructed plasmid, pBac[3×P3-DsRed2afm]-BmPheRS_α_A450G (Figure 1a), was used for germline transformation. The newly

Figure 2. (a) Structures of Phe and its analogues used in this study. (b) The procedure used to evaluate the incorporation of Phe analogues into silk fibroin. Fifth instar larvae of H01 and WT were fed a synthetic diet containing or not containing Phe analogues. The analogues were added to the diet from the third day of fifth instar until the start of spinning. Three male larvae were used for each test condition and reared at 25 °C. Three days after the start of spinning, the cocoons were harvested, weighed, and stored at −20 °C until analysis. Whole cocoon proteins were dissolved in LiBr solution, separated by SDS-PAGE and then subjected to reduction of disulfide linkages. Three protein components of fibroin, a fibroin heavy chain (FibH; ∼350 kDa), a fibroin light chain (FibL) (∼26 kDa), and a glycoprotein P25 (∼30 kDa), were detected on SDS-PAGE. The actual analysis gave multiple bands of sericins as minor components (see Figures 3 and 6), but they are omitted here for simplification. Stained bands of FibL were excised and in-gel digested with trypsin, and the resulting peptide fragments were subjected to MALDI-TOFMS analysis (see Figure S1 for peptide mass fingerprinting analysis of FibL) in order to detect, as a mass shift, the replacement of Phe by their analogues in FibL.

Figure 1. Germline transformation of B. mori. (a) Plasmid map of a piggyBac vector used for germline transformation of B. mori to generate the H01 transgenic line. The gene encoding the A450G mutant of the BmPheRS α-subunit was inserted downstream of the FibL promoter for specific expression in PSG. Expression of DsRed2 during the embryo stage was used to screen transgenic individuals. (b) The fifth instar larvae of the newly generated H01 transgenic line. (c) Verification of the specific expression of the gene encoding the A450G mutant of the BmPheRS α-subunit in PSG by RT-PCR. cDNA fragments of the A450G mutant and the wild-type of the BmPheRS αsubunit were amplified using independent primer sets after RT. The results of the negative control experiment without RT are shown in the top panel. Total RNA extracted from each silk gland component (ASG, MSG, and PSG) of the fifth instar larvae of H01 was used as a template.

incorporation of Phe analogues into silk fibroin. We reared fifth instar larvae of the H01 and wild-type (WT) strains on a synthetic diet (Table S1) containing or not containing Phe analogues. Since Phe is an essential amino acid for B. mori, the internal Phe concentration could be controlled by changing the amount of Phe in the synthetic diet. In their fifth instar, the H01 larvae grew normally in the manner of the WT larvae, even when ClPhe (in a 1/2 mol equiv to Phe) was added to the diet, and started spinning (Figure 3a). To measure the weight of fibroin produced under each condition, small pieces of each individual harvested cocoon were degummed with 8 M urea to remove the sericin layer without causing hydrolysis of fibroin.40 The weight of fibroin decreased for both H01 and WT by ClPhe administration, where the extent of decrease was significantly greater for H01: the fibroin production, respectively, decreased about 66 and 34% for H01 and WT (Figure 3b). The SDS-PAGE of whole cocoon proteins exhibited a decrease of fibroin relative to sericins when ClPhe was fed to H01 larvae (Figure 3c, the leftmost lane). On the other hand, the protein compositions and their molecular weights remained unchanged. We expected that the relative decrease of fibroin production was due to the ClPhe incorporation in protein biosynthesis in PSG cells, because a similar decrease of protein

generated transgenic line expressing the A450G mutant of the BmPheRS α-subunit specifically in PSG was named H01 (Figure 1b). Partial cDNA sequences of the transgene and its intrinsic wild-type counterpart were amplified by RT-PCR from ASG, MSG, and PSG-derived total RNA (Figure 1c), which verified the specific expression of the transgene in PSG. Incorporation Assay of ClPhe. Among the Phe analogues we have previously investigated using B. mori cultured cells, ClPhe (Figure 2a) appeared to exhibit the highest efficiency of incorporation into a reporter protein.33,34 We thus first administered ClPhe to fifth instar larvae of H01 to determine the effectiveness of the same methodology at an individual level. Figure 2b describes the procedure used to evaluate the 2684

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biosynthesis has been observed in previous experiments using cultured cells.33,34 MALDI-TOF-MS analysis of FibL after ingel digestion with trypsin confirmed the partial replacement of Phe by ClPhe (Figure 4). Each fragment peak accompanied a +34 Da counterpart, which matches the replacement of hydrogen (1H) by chlorine (35Cl). The peaks arising from the stable isotope, 37Cl, were overlapped at the +36 Da positions. A double incorporation peak was also observed in the case of the SGNFAGFR fragment. These observations demonstrated that ClPhe was successfully incorporated into B. mori silk fibroin without inducing any detrimental effects on the larval growth. Incorporation Assay of BrPhe. Our previous experiments using cultured cells demonstrated that BrPhe was incorporated into protein biosynthesis only when the concentration of Phe in the medium was lowered.34 When BrPhe was administered to the H01 larvae, incorporation of BrPhe was verified by the appearance of a +78 Da peak on mass analysis corresponding to the replacement of hydrogen (1H) by bromine (79Br; Figure 5a). The peak arising from the stable isotope, 81Br, was overlapped at the +80 Da position. Lowering the Phe content in the diet to ×0.5 the standard amount resulted in a remarkable increase in BrPhe incorporation (Figure 5a) but decreased fibroin production at the same time (Figure 5b). A similar inverse relationship between UAA incorporation and protein production was also observed in the previous experiments using cultured cells.34 Incorporation Assay of AzPhe. The success of ClPhe and BrPhe incorporation into silk fibroin prompted us to investigate the incorporation of AzPhe (Figure 2a), because it possesses an azide group that is highly useful for bio-orthogonal ligation reactions such as azide−alkyne cycloaddition (click chemistry) and Staudinger ligation.44 The introduction of such selective chemical handles would be quite useful for adding new properties to silk fibroin as demanded for specific applications. The previous experiments using cultured cells indicated that incorporation of AzPhe requires a significant reduction in the Phe concentration in culture media.34 We, therefore, evaluated the effect of lowering the amount of Phe in the diet on AzPhe incorporation. We prepared synthetic diets containing a constant amount of AzPhe (a 1/2 mol equiv to Phe in the standard diet composition) but varied amounts of Phe (×1.0, ×0.5, ×0.4,

Figure 3. Administration of ClPhe to the H01 and WT larvae. (a) The growth curves of the H01 and WT larvae. A synthetic diet with (+) or without (−) ClPhe (in a 1/2 mol equiv to Phe) was fed to the larvae from the third day of fifth instar until the start of spinning. (b) Fibroin production by the H01 and WT larvae. The weights of fibroin were calculated from the weights of whole cocoons and the weight loss by urea degumming. (c) Representative SDS-PAGE pattern of solutions in which constant amounts of whole cocoon proteins were dissolved. Separated proteins were stained with CBB, and FibH and FibL bands were labeled. The nonlabeled bands were sericins. P25 was not clearly observed here. The sericin content (%) in the cocoons estimated by urea degumming is shown above the gel image for each condition. All the numerical data are the average values of three independent experiments where each experiment employs three male larvae. The error bars in (a) and (b) represent standard deviations.

Figure 4. Detection of ClPhe incorporation in silk fibroin by MALDI-TOF-MS analysis of peptide fragments from in-gel digestion of FibL. Enlarged views are given for the regions around peaks assigned to Phe-containing fragments (see Figure S1), SGNFAGFR ([M + H]+ = 855.41 Da), QSLGPFFGHVGQNLNLINQLVINPGQLR ([M + H]+ = 3073.67 Da), and NSQSNNIAAYITAHLLPPVAQVFHQSAGSITDLLR ([M + H]+ = 3746.96 Da). The peak intensities at about 855, 3074, and 3748 Da were standardized in all spectra. The peaks marked with the asterisks are −17 Da (loss of NH2) counterparts of about 3074 Da peaks, which were formed via pyroglutamate formation of N-terminal glutamate.43 2685

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Figure 5. Incorporation of BrPhe into silk fibroin. The H01 larvae were fed a diet containing a BrPhe (in a 1/2 mol equiv to Phe) from the third day of fifth instar until the start of spinning. Phe content in the diet was set to be ×1.0 (standard) or ×0.5. (a) Representative MALDI-TOF-MS spectra of in-gel digested FibL with trypsin. An enlarged view of the region around a peak assigned to the SGNFAGFR fragment ([M + H]+ = 855.41 Da) is shown. The peak intensities at about 855 Da were standardized. (b) Weights of fibroin produced by the H01 larvae at Phe ×1.0 and ×0.5 in the presence of BrPhe.

×0.3, and ×0.2). Figure 6 shows the growth curves of the H01 and WT larvae fed diets. Lowering the Phe content in the diet to ×0.2 did not remarkably slow the growth of larvae without the addition of AzPhe (Figure 6a, open squares in magenta). However, when the H01 larvae were fed the Phe ×0.2 diet containing AzPhe, their growth appeared to be impeded (Figure 6a, closed squares in magenta) and their level of fibroin production was extremely low (Figure 6b, bar plots). In response to the decrease in the Phe amount in the diet, the fibroin weight gradually decreased (Figure 6b). SDS-PAGE of whole cocoon proteins exhibited a gradual decrease of FibH and FibL relative to sericins, along with a reduction in Phe in the diet, whereas the protein compositions and their molecular weights remained unchanged (Figure 6c). When the diet containing AzPhe with a × 0.2 amount of Phe was fed to H01 larvae, only about 5 mg fibroin/larva was harvested (Figure 6b). Under this condition, the larvae grew poorly (Figure 6a, closed squares in magenta) and made only thin cocoons with a sericin large content (Figure 6c). When WT larvae were reared under the same condition, they produced an approximately 7-fold greater amount of fibroin (Figure 6b). Although lowering the Phe content itself decreased the cocoon weight,45 the observed relative decrease of fibroin by AzPhe feeding strongly implied that AzPhe was specifically incorporated in protein biosynthesis in the PSG cells of H01 larvae. MALDI-TOF-MS analysis of the SGNFAGFR fragment from FibL showed that the relative intensity of a peak at the +15 Da position gradually increased with the decrease in the Phe amount in the diet. The observed mass increase corresponds to the replacement of hydrogen (1H) with an amino group (14N1H1H). No signals arising from unreduced azide groups (+41 Da) were observed (Figure 7a). It is known that aryl azides can be reduced to corresponding amines in some living organisms,46 in the presence of reducing agents,47 or by laser irradiation on mass analysis.48 Since the recognition of pamino- L -phenylalanine (AmPhe) with the recombinant BmPheRS A450G mutant was not observed in the previous in vitro experiment,34 it was not likely that AmPhe was directly incorporated into silk fibroin after in vivo reduction of AzPhe in

Figure 6. Administration of AzPhe to the H01 and WT larvae. (a) The growth curves of the H01 and WT larvae fed synthetic diets with (+) or without (−) AzPhe (a half molar equivalent to Phe in the standard diet composition) from the third day of fifth instar until the start of spinning. The amounts of Phe in the diet declined from ×1.0, ×0.5, ×0.4, ×0.3 to ×0.2 of the standard amount. (b) The weights of fibroin (bar plots) and the ratio of peak intensities (open circles; 870 Da peak/855 Da peak) in MALDI-TOF-MS spectra (Figure 7a) are shown against the Phe amount in the diet. The ratio of peak intensities was used as an index of the replacement of Phe with AzPhe. (c) Representative SDS-PAGE pattern of solutions in which constant amounts of whole cocoon proteins were dissolved. Separated proteins were stained with CBB, and FibH, FibL, and P25 bands were labeled. The nonlabeled bands were sericins. The sericin content (%) estimated by urea degumming is shown above the gel image for each condition. All the numerical data are the average values of three independent experiments, where each experiment employed three male larvae. The error bars in (a) and (b) represent standard deviations.

larvae. We thus speculated that AzPhe was successfully incorporated into silk fibroin but AzPhe was reduced to AmPhe by the reducing agent upon sample preparation for SDS-PAGE and laser irradiation on mass analysis. Neither +15 Da nor +41 Da peaks were clearly observed for the other Phecontaining fragments, probably due to the relatively lower incorporation of AzPhe than ClPhe. The ratio of peak intensities between the SGNFAGFR fragments and their +15 Da counterparts in MALDI-TOF-MS spectra (Figure 7a) was plotted against the Phe amount in the diet as an index of the replacement of Phe with AzPhe (Figure 6b, open circles). As expected, there was little incorporation of AzPhe in the presence of a standard amount of Phe in the diet. The ratio increased drastically when the Phe amount was lowered to ×0.3, but exhibited a large deviation under the ×0.2 Phe condition as well as an extremely small production of fibroin. These observations indicated that a larger incorporation 2686

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Figure 7. Detection of AzPhe incorporation in silk fibroin. (a) Representative MALDI-TOF-MS spectra of in-gel digested FibL with trypsin. An enlarged view of the region around a peak assigned to the SGNFAGFR fragment ([M + H]+ = 855.41 Da) is shown. The peak intensities at about 855 Da were standardized in all spectra. (b) Click reactions on AzPhe-incorporated silk fibroin after urea degumming. AzPhe-incorporated silk fibroin was reacted with alkyne-PEG4-biotin or alkyne-PEG3-carboxyrhodamine 110 to yield click-modified silk fibroins. After the reaction with alkyne-PEG4-biotin in homogeneous solution, biotin molecules were detected by Western blotting using HRP-streptavidin. After the reaction with alkyne-PEG3-carboxyrhodamine 110 on fiber, green fluorescence from the fluorophores (carboxyrhodamine 110) was observed with a fluorescence stereomicroscope by 480 nm excitation. Fluorescent images were merged with bright field images.

of AzPhe requires a lower Phe content in the diet, but that the growth of larvae is also impeded under this condition. Azide−Alkyne Cycloaddition Reactions on Silk Fibroin. The incorporation of AzPhe in silk fibroin was further confirmed by click reactions between the azide groups in silk fibroin and labeling reagents bearing terminal alkyne groups (Figure 7b). Part of the cocoon was first degummed with 8 M urea to remove the sericin layer, then dissolved in LiBr aqueous solution, and reacted with alkyne-PEG4-biotin in the presence of a Cu catalyst. Biotin molecules attached to fibroin were then detected by Western blotting. Signals from biotin were clearly detected on FibH, FibL, and P25, confirming the incorporation of AzPhe into silk fibroin (Figure 7b). Urea-degummed fibroin was also reacted with alkyne-PEG3-carboxyrhodamine 110 in its native fibrous state. Green fluorescence from the appended fluorophores was clearly observed (Figure 7b). These results demonstrated that external functional molecules can be easily attached to silk fibroin using highly selective click reactions under not only homogeneous, but also heterogeneous reaction conditions. These data also demonstrated that the extent of derivatization could be controlled by controlling the AzPhe content in fibroin, that is, by changing the conditions under which the larvae were reared. Optimization of AzPhe Dosage. To incorporate a detectable amount of AzPhe into silk fibroin, the amount of Phe should not be greater than ×0.3 the standard amount (Figure 7a). However, under the condition of Phe ×0.3, the fibroin production was reduced to about 21 mg/larva, which is approximately 1/5 the normal fibroin production without UAAs (Figure 3b). Such low productivity of AzPheincorporated silk fibroin would become a major constraint for its practical applications. We thus further optimized the molar equivalent of AzPhe added to the diet under the fixed Phe content of ×0.3. Figure 8 shows the relationships among AzPhe dosage (from 0.75 to 0.05 mol equiv to the standard amount of Phe), fibroin production, and the ratio of peak intensities as an

Figure 8. Dependence of fibroin production and AzPhe incorporation on AzPhe dosage. Weights of the produced fibroin (bar plots) and the ratio of peak intensities (open circles; 870 Da peak/855 Da peak) in MALDI-TOF-MS analysis are shown against the amount of Phe added to the diet from the third day of fifth instar until the start of spinning. The ratio of peak intensities was used as an index of the replacement of Phe with AzPhe. The amount of AzPhe was shown as the molar equivalent to Phe in the standard diet composition. The Phe content in the diet was fixed at ×0.3. All data are the average values of three independent experiments, where each experiment employed three male larvae. The error bars represent standard deviations.

index of AzPhe incorporation. Compared with the initial condition (0.5 mol equiv of AzPhe), the fibroin production decreased with a larger dosage of AzPhe (0.75 mol equiv). In contrast, fibroin production gradually increased when the AzPhe dosage decreased. The ratio of peak intensities was almost unchanged in the range from a 0.15 to 0.75 mol equiv, whereas it was remarkably decreased below a 0.15 mol equiv. 2687

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From these experiments, we established that the optimum condition for producing AzPhe-incorporated silk fibroin was a 0.15 mol equiv of the AzPhe dosage at the Phe content of ×0.3. The intensity of the about 870 Da peak, where one of the two Phe residues was replaced with AzPhe (observed in a reduced form), was about 3% of the intensity of its parental peak at about 855 Da at the optimized condition (0.15 equiv of AzPhe at the Phe content of ×0.3; Figure 8). Under the assumption that the ionization efficiencies of these two peptide fragments were the same, the incorporation rate of AzPhe was roughly estimated to be 1.5% per one Phe residue. The replacement of Phe to AzPhe was expected to take place equally at all Phe positions. Silk fibroin can be regarded as a heterodimer of FibH and FibL,49 which respectively contain 28 and 7 Phe residues in their matured forms.38,50 Under these assumptions, the 1.5% replacement of Phe to AzPhe leads to the estimation that approximately half of fibroin heterodimers possess one AzPhe residue. If click reactions on AzPhe proceed with high efficiency, the estimated replacement rate would be adequate for adding novel characteristics to silk fibroin, as suggested by the model reactions shown in Figure 7. Stability of Azide Groups on Standard Alkaline Degumming. When B. mori silk is used in biomedical applications, the sericin layer should be completely removed, because impurities from the sericin layer might cause inflammatory responses.13,51 In this study, a urea degumming method was employed to estimate the sericin content in cocoons because this method does not cause hydrolysis of fibroin molecules40 and, thus, does not restrict the subsequent analyses. Click reactions with urea-degummed silk fibroin proved that azide groups tolerate the urea degumming process (Figure 7b). However, more intensive degumming in boiling Na2CO3 solution was commonly employed as the most approved method for complete sericin removal.3 In contrast to the relatively gentle urea degumming, Na2CO3 degumming causes hydrolysis of fibroin molecules.40 We thus finally tested the stability of azide groups on this standard Na 2 CO 3 degumming for the future application of AzPhe-incorporated silk fibroin in the biomedical fields. For this purpose, we produced cocoons under an optimized AzPhe administration condition (0.15 equiv of AzPhe at the Phe content of ×0.3; Figure 8). The harvested cocoons were degummed in boiling Na2CO3 to give Na2CO3-degummed silk (Figure 9a). Part of the Na2CO3-degummed silk was dissolved in LiBr solution and reacted with alkyne-PEG4-biotin, as described above, followed by Western blotting analysis to assess the survival of the azide groups (Figure 9b). The AzPhe-incorporated silk fibroin degummed in Na2CO3 gave an SDS-PAGE pattern identical to the normal one when stained with CBB: a faint FibL band was observed over a smeared background (Figure 9b, left panel). The smeared background resulted from hydrolysis of FibH.40 Western blotting with HRP-streptavidin gave distinct signals only for the AzPhe-incorporated silk fibroin (Figure 9b, right panel). This result clearly showed that azide groups survived the Na2CO3 degumming process and selectively reacted with alkyne-PEG4-biotin. The observed stability of azide groups against the most approved degumming condition indicated that the AzPhe-incorporated silk could be used as a versatile platform to prepare “clickable” silk materials in various forms via the established processing techniques.3 Das et al. recently reported the preparation of azidefunctionalized silk fibroin based on diazonium coupling

Figure 9. Detection of azides after standard alkaline degumming in boiling Na2CO3. (a) The cocoons obtained by AzPhe administration to the H01 larvae under the optimized condition (0.15 mol equiv of AzPhe at Phe ×0.3) were degummed in boiling 0.02 M Na2CO3 for 30 min. The Na2CO3-degummed silk fibroin was dissolved in 8 M LiBr and then reacted with alkyne-PEG4-biotin. (b) Normal and AzPheincorporated (+AzPhe) silk fibroin after Na2CO3 degumming were separated by SDS-PAGE and stained with CBB (left panel). The same samples were transferred to a PVDF membrane, and biotin molecules attached to fibroin were detected with HRP-streptavidin (right panel).

chemistry and subsequent derivatization with click reactions.52 More recently, Galeotti et al. reported a similar approach in which they modified the amino and hydroxyl groups in silk fibroin with an azide-bearing cyanuric acid derivative.53 These studies demonstrate the versatility of click chemistry for the preparation of silk materials with characteristics tuned for specific applications. Our approach described herein enables in vivo production of azide-functionalized silk fibroin in its natural fibrous form without the use of any chemical treatments. This feature would provide tremendous advantages for future applications, since it would permit the biological production of azide-functionalized silk in a clean (without chemical reactions), easy (requiring only feeding to larvae), and selective manner. The word “selective” here means that our approach can regulate the introduction of azide groups strictly at the positions of Phe residues in silk fibroin, although their replacement with AzPhe occurs randomly at each Phe position, probably with uniform probability.

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CONCLUSION Here we have reported the first application of residue-specific UAA mutagenesis of proteins to B. mori larvae. Partial replacement of Phe residues in silk fibroin with three kinds of their unnatural analogues, ClPhe, BrPhe, and AzPhe, was 2688

dx.doi.org/10.1021/bm5005349 | Biomacromolecules 2014, 15, 2682−2690

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(10) Lovett, M. L.; Cannizzaro, C. M.; Vunjak-Novakovic, G.; Kaplan, D. L. Biomaterials 2008, 29, 4650−4657. (11) Kameda, T.; Hashimoto, T.; Tamada, Y. J. Mater. Sci. 2011, 46, 7923−7930. (12) Mandal, B. B.; Grinberg, A.; Seok Gil, E.; Panilaitis, B.; Kaplan, D. L. Proc. Natl. Acad. Sci. U.S.A. 2012, 109, 7699−7704. (13) Leal-Egaña, A.; Scheibel, T. Biotechnol. Appl. Biochem. 2010, 55, 155−167. (14) Tamura, T.; Thibert, C.; Royer, C.; Kanda, T.; Eappen, A.; Kamba, M.; Komoto, N.; Thomas, J.-L.; Mauchamp, B.; Chavancy, G.; Shirk, P.; Fraser, M.; Prudhomme, J.-C.; Couble, P. Nat. Biotechnol. 2000, 18, 81−84. (15) Teulé, F.; Miao, Y.-G.; Sohn, B.-H.; Kim, Y.-S.; Hull, J. J.; Fraser, M. J.; Lewis, R. V.; Jarvis, D. L. Proc. Natl. Acad. Sci. U.S.A. 2012, 109, 923−928. (16) Kuwana, Y.; Kojima, K.; Tamada, Y. J. Silk Sci. Technol. Jpn. 2012, 20, 81−83. (17) Hino, R.; Tomita, M.; Yoshizato, K. Biomaterials 2006, 27, 5715−5724. (18) Kambe, Y.; Yamamoto, K.; Kojima, K.; Tamada, Y.; Tomita, N. Biomaterials 2010, 31, 7503−7511. (19) Nagano, A.; Tanioka, Y.; Sakurai, N.; Sezutsu, H.; Kuboyama, N.; Kiba, H.; Tanimoto, Y.; Nishiyama, N.; Asakura, T. Acta Biomater. 2011, 7, 1192−1201. (20) Tomita, M. Biotechnol. Lett. 2011, 33, 645−654. (21) Wang, F.; Xu, H.; Yuan, L.; Ma, S.; Wang, Y.; Duan, X.; Duan, J.; Xiang, Z.; Xia, Q. Transgenic Res. 2013, 22, 925−938. (22) Murphy, A. R.; Kaplan, D. L. J. Mater. Chem. 2009, 19, 6443− 6450. (23) Chin, J. W. Annu. Rev. Biochem. 2014, 83, 379−408. (24) Singh-Blom, A.; Hughes, R. A.; Ellington, A. D. In Enzyme Engineering: Methods and Protocols, Methods in Molecular Biology; Samuelson, J. C., Ed.; Humana Press: Totowa, NJ, 2013; Vol. 978, pp 93−114. (25) Wiltschi, B.; Budisa, N. Appl. Microbiol. Biotechnol. 2007, 74, 739−753. (26) Ngo, J. T.; Tirrell, D. A. Acc. Chem. Res. 2011, 44, 677−685. (27) Wang, L.; Brock, A.; Herberich, B.; Schultz, P. G. Science 2001, 292, 498−500. (28) Chin, J. W.; Cropp, T. A.; Anderson, J. C.; Mukherji, M.; Zhang, Z.; Schultz, P. G. Science 2003, 301, 964−967. (29) Link, A. J.; Tirrell, D. A. Methods 2005, 36, 291−298. (30) Bianco, A.; Townsley, F. M.; Greiss, S.; Lang, K.; Chin, J. W. Nat. Chem. Biol. 2012, 8, 748−750. (31) Greiss, S.; Chin, J. W. J. Am. Chem. Soc. 2011, 133, 14196− 14199. (32) Parrish, A. R.; She, X.; Xiang, Z.; Coin, I.; Shen, Z.; Briggs, S. P.; Dillin, A.; Wang, L. ACS Chem. Biol. 2012, 7, 1292−1302. (33) Teramoto, H.; Kojima, K.; Kajiwara, H.; Ishibashi, J. ChemBioChem 2012, 13, 61−65. (34) Teramoto, H.; Kojima, K. J. Insect Biotechnol. Sericol. 2013, in press (online version is available at http://jsss.or.jp/modules/ d3downloads/index.php?page=visit&cid=8&lid=59). (35) Arai, N.; Ito, T. J. Sericult. Sci. Jpn. 1964, 33, 107−110. (36) Ibba, M.; Kast, P.; Hennecke, H. Biochemistry (Moscow) 1994, 33, 7107−7112. (37) Kotik-Kogan, O.; Moor, N.; Tworowski, D.; Safro, M. Structure 2005, 13, 1799−1807. (38) Zhou, C.-Z.; Confalonieri, F.; Medina, N.; Zivanovic, Y.; Esnault, C.; Yang, T.; Jacquet, M.; Janin, J.; Duguet, M.; Perasso, R.; Li, Z.-G. Nucleic Acids Res. 2000, 28, 2413−2419. (39) Sato, M.; Kojima, K.; Sakuma, C.; Murakami, M.; Aratani, E.; Takenouchi, T.; Tamada, Y.; Kitani, H. PLoS One 2012, 7, e34632. (40) Yamada, H.; Nakao, H.; Takasu, Y.; Tsubouchi, K. Mater. Sci. Eng., C 2001, 14, 41−46. (41) Niedermeyer, T. H. J.; Strohalm, M. PLoS One 2012, 7, e44913. (42) Sehnal, F.; Sutherland, T. Prion 2008, 2, 145−153. (43) Thiede, B.; Lamer, S.; Mattow, J.; Siejak, F.; Dimmler, C.; Rudel, T.; Jungblut, P. R. Rapid Commun. Mass Spectrom. 2000, 14, 496−502.

accomplished in this study. ClPhe and BrPhe were incorporated into silk fibroin simply by adding them to the diet, whereas incorporation of AzPhe required cutting down the Phe content in the diet. From the practical point of view, the success of AzPhe incorporation is quite significant due to the utility of azide groups for bio-orthogonal ligation reactions. In fact, azide groups on silk fibroin were active for azide−alkyne cycloaddition reactions (click chemistry). It should be noted that the reaction proceeded not only in a solubilized state but also in a solid (fibrous) state, which indicates that any silk-based materials could be easily modified after they are prepared and even just before use. In addition, azide groups in silk fibroin were stable in the standard alkaline degumming process necessary for complete removal of the sericin layer, demonstrating that the conventional processing techniques can be directly applied to the AzPhe-incorporated silk fibroin. Moreover, AzPhe could be available as an active site for photochemistry.44 Our findings that UAA mutagenesis can be successfully performed for silk proteins produced by B. mori will further enhance the importance of this organism for industrial applications.

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ASSOCIATED CONTENT

S Supporting Information *

Supplementary experimental procedures for piggyBac vector construction, RT-PCR, preparation of the synthetic diet, Western blotting, and peptide mass fingerprinting of FibL. Table S1: Standard composition of the synthetic diet for B. mori. Figure S1: Identification of FibL by peptide mass fingerprinting analysis. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: teramoto@affrc.go.jp. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We acknowledge the Transgenic Silkworm Research Unit at NIAS for providing silkworm eggs. We thank Dr. J. Ishibashi for help with the mass analysis and Dr. Q. Zhang and Ms. M. Murakami for their technical assistance. This work was supported by JSPS KAKENHI Grant Number 24688008.

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