Genetic hybridization of highly active exogenous functional proteins into silk-based materials using “light-clothing” strategy

Abstract

•Exogenous proteins are genetically hybridized into silk materials•Trimming redundant parts of additive domains enhances exogenous protein activity•This trim does not compromise the genetic hybridization process Bio-synthesis of recombinant proteins using transgenic silkworms provides a promising bottom-up approach to design and functionalize silk materials. To ensure successful genetic hybridization, protein(s) of interest (POI) are routinely fused with additive domains from natural silk to form fusion POI (F-POI). However, the additive domains often lead to superfluous post-translational modifications and abnormal protein folding, thus severely impairing F-POI. Here, a “light-clothing” strategy, i.e., rationally trimming redundant parts of additive domains, is demonstrated to increase the structural similarity between F-POI and native POI, and, therefore, the activity of F-POI. The silk materials functionalized with highly active F-POI, such as growth factors, can be produced further as biomaterials for tissue regeneration. This strategy provides a solution to overcome the major technical hurdle of using transgenic silkworms as a bio-synthesis platform to develop high-performance materials. Genetic hybridization of exogenous functional protein(s) of interest (POI) with silk using transgenic silkworms provides promising bio-synthesis platforms to design silk-based materials. To ensure successful hybridization, additive domains from natural silk are routinely fused with POI to form fusion POI (F-POI), but they often lead to superfluous post-translational modifications, compromise normal folding, and severely impair the activity of POI. Here, “light-clothing” strategy, i.e., rationally trimming redundant parts of additive domains, is proposed. As visualized by green fluorescent protein as marker POI, this strategy avoids supernumerary post-translational modifications in F-POI synthesis, and enables its normal secretion and extraction. Applying epidermal growth factor as model functional POI, material fabrication versatility of the extracted mixture of F-POI and silk is demonstrated. This strategy increases structural similarity between F-POI and native POI and remarkably enhances F-POI activity to the same level as commercial POI, which facilitates applications of these advanced bio-synthesis platforms for developing high-performance materials. Genetic hybridization of exogenous functional protein(s) of interest (POI) with silk using transgenic silkworms provides promising bio-synthesis platforms to design silk-based materials. To ensure successful hybridization, additive domains from natural silk are routinely fused with POI to form fusion POI (F-POI), but they often lead to superfluous post-translational modifications, compromise normal folding, and severely impair the activity of POI. Here, “light-clothing” strategy, i.e., rationally trimming redundant parts of additive domains, is proposed. As visualized by green fluorescent protein as marker POI, this strategy avoids supernumerary post-translational modifications in F-POI synthesis, and enables its normal secretion and extraction. Applying epidermal growth factor as model functional POI, material fabrication versatility of the extracted mixture of F-POI and silk is demonstrated. This strategy increases structural similarity between F-POI and native POI and remarkably enhances F-POI activity to the same level as commercial POI, which facilitates applications of these advanced bio-synthesis platforms for developing high-performance materials. During the past decades, silks, produced by sericulture or bioengineered silk via fermentation, have expanded their applicability from the millennia-old textile industry to various emerging areas, such as electronics, photonics, energy, environmental science, and biomedicine.1Huang W. Ling S. Li C. Omenetto F.G. Kaplan D.L. Silkworm silk-based materials and devices generated using bio-nanotechnology.Chem. Soc. Rev. 2018; 47: 6486-6504Google Scholar Genetic hybridization of exogenous protein(s) of interest (POI) into silk-based materials using transgenic silkworm (Bombyx mori) as a higher eukaryotic bio-hybridization platform provides a promising bottom-up approach to design, synthesize, and functionalize these materials.2Iizuka T. Sezutsu H. Tatematsu K. Kobayashi I. Yonemura N. Uchino K. Nakajima K. Kojima K. Takabayashi C. Machii H. et al.Colored fluorescent silk made by transgenic silkworms.Adv. Funct. Mater. 2013; 23: 5232-5239Google Scholar, 3Tomita M. Munetsuna H. Sato T. Adachi T. Hino R. Hayashi M. Shimizu K. Nakamura N. Tamura T. Yoshizato K. Transgenic silkworms produce recombinant human type III procollagen in cocoons.Nat. Biotechnol. 2003; 21: 52-56Google Scholar, 4Kurihara H. Sezutsu H. Tamura T. Yamada K. Production of an active feline interferon in the cocoon of transgenic silkworms using the fibroin H-chain expression system.Biochem. Biophys. Res. Commun. 2007; 355: 976-980Google Scholar, 5Saotome T. Hayashi H. Tanaka R. Kinugasa A. Uesugi S. Tatematsu K.-i. Sezutsu H. Kuwabara N. Asakura T. Introduction of VEGF or RGD sequences improves revascularization properties of Bombyx mori silk fibroin produced by transgenic silkworm.J. Mater. Chem. B. 2015; 3: 7109-7116Google Scholar The transgenic silkworm techniques hold exciting opportunities to combine recent progress in modern bioengineering with advantages of traditional farming, i.e., production of proteins at a large scale in a cost-effective, non-toxic and eco-friendly way.6Leem J.W. Fraser M.J. Kim Y.L. Transgenic and diet-enhanced silk production for reinforced biomaterials: a metamaterial perspective.Annu. Rev. Biomed. Eng. 2020; 22: 79-102Google Scholar Compared with prokaryotic bio-hybridization platforms (e.g., Escherichia coli), the transgenic silkworm has a much more versatile capacity to synthesize complex POI by (1) assembling subunit complexes of POI; (2) avoiding the formation of POI inclusion body; and (3) enabling essential post-translational modifications of POI, such as signal peptide excision, phosphorylation, glycosylation, and disulfide bond formation.6Leem J.W. Fraser M.J. Kim Y.L. Transgenic and diet-enhanced silk production for reinforced biomaterials: a metamaterial perspective.Annu. Rev. Biomed. Eng. 2020; 22: 79-102Google Scholar Moreover, the transgenic silkworm does not produce endotoxins7Gorbet M.B. Sefton M.V. Endotoxin: the uninvited guest.Biomaterials. 2005; 26: 6811-6817Google Scholar and is able to hybridize POI into natural silk with long repeat sequences and high molecular weight. Those advantages are difficult to achieve by bioengineered fermentation.8Huang W. Ebrahimi D. Dinjaski N. Tarakanova A. Buehler M.J. Wong J.Y. Kaplan D.L. Synergistic integration of experimental and simulation approaches for the de novo design of silk-based materials.Acc. Chem. Res. 2017; 50: 866-876Google Scholar Since the transgenic silkworm technique was developed in 2003,3Tomita M. Munetsuna H. Sato T. Adachi T. Hino R. Hayashi M. Shimizu K. Nakamura N. Tamura T. Yoshizato K. Transgenic silkworms produce recombinant human type III procollagen in cocoons.Nat. Biotechnol. 2003; 21: 52-56Google Scholar many studies have used this advanced bio-synthesis platform to genetically engineer silk-based materials (Table S1). Essentially, the POI synthesized by transgenic silkworms are fusion proteins, i.e., fusion POI (F-POI). As a conventional strategy, additional structural domains derived from endogenic natural silk proteins are routinely introduced upstream and/or downstream of the structural sequences of native POI to form an F-POI. In this way, synthesis and secretion of F-POI and natural silk into cocoons proceeds simultaneously.3Tomita M. Munetsuna H. Sato T. Adachi T. Hino R. Hayashi M. Shimizu K. Nakamura N. Tamura T. Yoshizato K. Transgenic silkworms produce recombinant human type III procollagen in cocoons.Nat. Biotechnol. 2003; 21: 52-56Google Scholar,4Kurihara H. Sezutsu H. Tamura T. Yamada K. Production of an active feline interferon in the cocoon of transgenic silkworms using the fibroin H-chain expression system.Biochem. Biophys. Res. Commun. 2007; 355: 976-980Google Scholar However, specific parts of the additional structural domains can lead to superfluous post-translational modifications of F-POI and compromise its normal folding.4Kurihara H. Sezutsu H. Tamura T. Yamada K. Production of an active feline interferon in the cocoon of transgenic silkworms using the fibroin H-chain expression system.Biochem. Biophys. Res. Commun. 2007; 355: 976-980Google Scholar,5Saotome T. Hayashi H. Tanaka R. Kinugasa A. Uesugi S. Tatematsu K.-i. Sezutsu H. Kuwabara N. Asakura T. Introduction of VEGF or RGD sequences improves revascularization properties of Bombyx mori silk fibroin produced by transgenic silkworm.J. Mater. Chem. B. 2015; 3: 7109-7116Google Scholar,9Zhao A. Zhao T. Zhang Y. Xia Q. Cheng L. Zhou Z. Xiang Z. Nakagaki M. New and highly efficient expression systems for expressing selectively foreign protein in the silk glands of transgenic silkworm.Transgenic Res. 2010; 19: 29-44Google Scholar,10Long D. Lu W. Zhang Y. Guo Q. Xiang Z. Zhao A. New insight into the mechanism underlying fibroin secretion in silkworm, Bombyx mori.FEBS J. 2015; 282: 89-101Google Scholar These abnormalities have been demonstrated to often result in the very low (e.g., 0.9% of the commercially available POI)11Hino R. Tomita M. Yoshizato K. The generation of germline transgenic silkworms for the production of biologically active recombinant fusion proteins of fibroin and human basic fibroblast growth factor.Biomaterials. 2006; 27: 5715-5724Google Scholar or no activity4Kurihara H. Sezutsu H. Tamura T. Yamada K. Production of an active feline interferon in the cocoon of transgenic silkworms using the fibroin H-chain expression system.Biochem. Biophys. Res. Commun. 2007; 355: 976-980Google Scholar,12Kambe Y. Kojima K. Tamada Y. Tomita N. Kameda T. Silk fibroin sponges with cell growth-promoting activity induced by genetically fused basic fibroblast growth factor.J. Biomed. Mater. Res. A. 2016; 104: 82-93Google Scholar of F-POI, which seriously limits the practical applications of this advanced bio-synthesis platform for developing high-performance silk-based materials, in particular in the biomedical field.13Long D. Xiao B. Merlin D. Genetically modified silk fibroin nanoparticles for drug delivery: preparation strategies and application prospects.Nanomedicine. 2020; 15: 1739-1742Google Scholar Therefore, this study aimed to enhance F-POI activity via rationally trimming redundant parts of the additional structural domains without compromising the genetic hybridization process (i.e., synthesis, secretion, extraction, and fabrication) into silk-based materials (Figure 1A). We termed this trimming process the “light-clothing” strategy as the trimmed F-POI is not over-encumbered with additional structural domains, whereas the conventional (i.e., untrimmed) synthesis for F-POI was given the term “heavy-clothing” strategy. As a proof of concept, transgenic silkworms were used to genetically hybridize growth factors as functional POI into silk fibroin (SF)-based materials for tissue engineering applications by using this light-clothing strategy. SF, the most commonly used silk protein exacted from B. mori cocoons, was selected since this protein attracts a mounting interest devoted to tissue engineering applications due to its excellent biocompatibility, robust mechanical properties, hypoallergenic features, and tunable biodegradation.14Cheng X. Deng D. Chen L. Jansen J.A. Leeuwenburgh S.G.C. Yang F. Electrodeposited assembly of additive-free silk fibroin coating from pre-assembled nanospheres for drug delivery.ACS Appl. Mater. Interfaces. 2020; 12: 12018-12029Google Scholar, 15Han C. Yao Y. Cheng X. Luo J. Luo P. Wang Q. Yang F. Wei Q. Zhang Z. Electrophoretic deposition of gentamicin-loaded silk fibroin coatings on 3D-printed porous cobalt–chromium–molybdenum bone substitutes to prevent orthopedic implant infections.Biomacromolecules. 2017; 18: 3776-3787Google Scholar, 16Cheng X. Yang F. More than just a barrier—challenges in the development of guided bone regeneration membranes.Matter. 2019; 1: 558-560Google Scholar, 17Umuhoza D. Yang F. Long D. Hao Z. Dai J. Zhao A. Strategies for tuning the biodegradation of silk fibroin-based materials for tissue engineering applications.ACS Biomater. Sci. Eng. 2020; 6: 1290-1310Google Scholar In this study, we first used enhanced green fluorescent protein (GFP) as a marker POI for visualization purposes. By trimming specific redundant parts of the additional structural domains of F-POI, the specific effects of this trimming on the synthesis, secretion, and extraction process of F-POI were investigated in detail. Subsequently, by selecting the human epidermal growth factor (EGF) as a functional model POI, we demonstrated the ease of fabricating a mixture of SF and F-POI into various tissue engineering scaffolds. The activity of this “lightly clothed” F-POI was compared with “heavily clothed” F-POI and native POI (i.e., a commercially available control). Furthermore, the underlying mechanism of the activity of lightly clothed F-POI was studied by theoretical modeling. Among various transgenic silk gland expression systems, SF heavy chain (SF-h) expression system was selected in this study to synthesize F-POI in view of its superior efficiency.9Zhao A. Zhao T. Zhang Y. Xia Q. Cheng L. Zhou Z. Xiang Z. Nakagaki M. New and highly efficient expression systems for expressing selectively foreign protein in the silk glands of transgenic silkworm.Transgenic Res. 2010; 19: 29-44Google Scholar,18Long D. Lu W. Zhang Y. Bi L. Xiang Z. Zhao A. An efficient strategy for producing a stable, replaceable, highly efficient transgene expression system in silkworm, Bombyx mori.Sci. Rep. 2015; 5: 8802Google Scholar The SF-h consists of three domains: an N-terminal domain (146 amino acids), a repetitive domain, and a C-terminal domain (57 amino acids) (Figure 1B). The conventional SF-h expression system aims to synthetize a heavily clothed F-POI by replacing the whole repetitive domain of SF-h with the original structural sequences of POI, where additional structural domains derived from SF-h (i.e., N- and C-terminal domains) are kept and fused (Figure 1B). SF-h is synthesized and secreted by the silk gland of the silkworm.19Inoue S. Tanaka K. Arisaka F. Kimura S. Ohtomo K. Mizuno S. 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. 2000; 275: 40517-40528Google Scholar A signal peptide, located on the N terminus, is essential for SF-h to allow for translocation across endoplasmic reticulum membrane, transportation from the silk gland cells into the lumen, and finally cleavage from the mature SF-h.9Zhao A. Zhao T. Zhang Y. Xia Q. Cheng L. Zhou Z. Xiang Z. Nakagaki M. New and highly efficient expression systems for expressing selectively foreign protein in the silk glands of transgenic silkworm.Transgenic Res. 2010; 19: 29-44Google Scholar,20Wang S. Guo T. Guo X. Huang J. Lu C. In vivo analysis of fibroin heavy chain signal peptide of silkworm Bombyx mori using recombinant baculovirus as vector.Biochem. Biophys. Res. Commun. 2006; 341: 1203-1210Google Scholar The signal peptide of heavily clothed F-POI was predicted by SignalP 5.0 software. The results showed that the 1st–21st amino acid residues on the additional N-terminal domain might act as its signal peptide (probability: 98.3%), and the relevant cleavage site of signal peptide might occur between the 21st and 22nd amino acids (probability: 75.5%) (Figures 1B and 1C; Table S2). Thus, we speculated that the 1st–21st amino acid residues on the additional N-terminal domain of heavily clothed F-POI (i.e., a shortened N-terminal domain) should be reserved in our strategy, although it could be cleaved during secretion. Moreover, a recent study found that the last of the 1st–20th amino acid residues on the C terminus of SF-h contains three crucial cysteine residues (1et, 4th, and 20th) (Figure 1B). These residues can form intra- or intermolecular disulfide bonds, thereby enabling correct folding of natural SF-h and assembly into fibroin units for secretion.21Hu W. Lu W. Wei L. Zhang Y. Xia Q. Molecular nature of dominant naked pupa mutation reveals novel insights into silk production in Bombyx mori.Insect Biochem. Mol. Biol. 2019; 109: 52-62Google Scholar Therefore, we speculated that the last of the 1st–20th amino acid residues on additional C-terminal domain of heavily clothed F-POI (i.e., a shortened C-terminal domain) should also be retained in our strategy based on trimming redundant parts of additive domains. In addition, we predicted potent post-translational modification sites on additional N- and C-terminal domains of heavily clothed F-POI (Table S2). We found many potential phosphorylation sites and a potential N-glycosylation site, which are all located outside the regions of shortened N- and C-terminal domains (Figures 1B, 1D–1F, S1, and S2). For bioengineered proteins, supernumerary post-translational modifications during the heavily clothed F-POI synthesis process can reduce their activity.4Kurihara H. Sezutsu H. Tamura T. Yamada K. Production of an active feline interferon in the cocoon of transgenic silkworms using the fibroin H-chain expression system.Biochem. Biophys. Res. Commun. 2007; 355: 976-980Google Scholar,5Saotome T. Hayashi H. Tanaka R. Kinugasa A. Uesugi S. Tatematsu K.-i. Sezutsu H. Kuwabara N. Asakura T. Introduction of VEGF or RGD sequences improves revascularization properties of Bombyx mori silk fibroin produced by transgenic silkworm.J. Mater. Chem. B. 2015; 3: 7109-7116Google Scholar,22Ganapathy M. Plants as bioreactors—a review.Adv. Tech. Biol. Med. 2016; 4: 161Google Scholar Consequently, we hypothesized that, except for speculated shortened N- and C-terminal domains regions, all other parts of additional N- and C-terminal domains of heavily clothed F-POI should be deleted to enhance the activity of F-POI. First, GFP was used as a marker POI to visualize the effect of our light-clothing strategy based on trimming redundant parts of additive domains on POI synthesis, secretion, and exaction. Five kinds of F-POI (GFP) with different terminal domains were designed and genetically hybridized into natural SF (Figures 2A and S4–S6; see supplemental experimental procedures for more details): (1) complete N- and C-terminal domains (NGC group); (2) only complete N-terminal domain (NG group); (3) complete N-terminal domain and shortened C-terminal domain (NGCs group); (4) only shortened C-terminal domain (GCs group); and (5) shortened N- and C-terminal domains (NsGCs group). The corresponding transgenic strains were constructed and identified (Figure S7, Tables S4 and S5; see supplemental experimental procedures for more details). Red fluorescence emission from DsRed-Express can be observed in the eyes of moths (adults) (Figures S4 and S8), demonstrating that all designed F-POI (GFP) gene expression cassettes were successfully inserted into the genomes of transgenic silkworms.10Long D. Lu W. Zhang Y. Guo Q. Xiang Z. Zhao A. New insight into the mechanism underlying fibroin secretion in silkworm, Bombyx mori.FEBS J. 2015; 282: 89-101Google Scholar,18Long D. Lu W. Zhang Y. Bi L. Xiang Z. Zhao A. An efficient strategy for producing a stable, replaceable, highly efficient transgene expression system in silkworm, Bombyx mori.Sci. Rep. 2015; 5: 8802Google Scholar The cocoons from the NGC, NG, NGCs, and NsGCs groups displayed strong green fluorescence, while wild-type (WT) and GCs cocoons did not exhibit fluorescence (Figure 2B). Correspondingly, no F-POI (GFP) bands could be detected from cocoons in the GCs and WT groups in the SDS-PAGE and western blot (Figure 2C). These results pointed out that the complete absence of the N-terminal domain could impede secretion of F-POI into cocoons. Furthermore, estimated from SDS-PAGE and western blot, the actual molecular weight of F-POI (GFP) for the NGC, NGCs, and NG groups were 57, 53, and 50 kDa, respectively (Figures 2C and 2D). These values were all approximately 10 kDa larger than their theoretical values. This increase of the actual molecular weight of F-POI (GFP) has also been reported in previous studies using the conventional SF-h expression system to produce F-POI,5Saotome T. Hayashi H. Tanaka R. Kinugasa A. Uesugi S. Tatematsu K.-i. Sezutsu H. Kuwabara N. Asakura T. Introduction of VEGF or RGD sequences improves revascularization properties of Bombyx mori silk fibroin produced by transgenic silkworm.J. Mater. Chem. B. 2015; 3: 7109-7116Google Scholar,9Zhao A. Zhao T. Zhang Y. Xia Q. Cheng L. Zhou Z. Xiang Z. Nakagaki M. New and highly efficient expression systems for expressing selectively foreign protein in the silk glands of transgenic silkworm.Transgenic Res. 2010; 19: 29-44Google Scholar,10Long D. Lu W. Zhang Y. Guo Q. Xiang Z. Zhao A. New insight into the mechanism underlying fibroin secretion in silkworm, Bombyx mori.FEBS J. 2015; 282: 89-101Google Scholar which can be attributed to the post-translational modifications on additional C- and N-terminal domains of F-POI (Figure 1B). In comparison, the actual molecular weight of F-POI (GFP) in the NsGCs group decreased from 53 to 29 kDa as compared with the molecular weight of F-POI (GFP) in the NGCs group, which was very close to its theoretical value (Figure 2D). This significant decrease indicates that the majority of superfluous post-translational modifications might occur on additional N-terminal domains other than the C-terminal domain. Moreover, the matching of actual and theoretical molecular weights in the NsGCs group suggests that superfluous post-translational modifications were successfully prevented by our light-clothing strategy. In addition, the theoretical molecular weights of F-POI (GFP) in the GCs and NsGCs groups were similar (Figure 2D) since the shortened N-terminal domain only contained a predicted signal peptide and would be cleaved during secretion.9Zhao A. Zhao T. Zhang Y. Xia Q. Cheng L. Zhou Z. Xiang Z. Nakagaki M. New and highly efficient expression systems for expressing selectively foreign protein in the silk glands of transgenic silkworm.Transgenic Res. 2010; 19: 29-44Google Scholar,20Wang S. Guo T. Guo X. Huang J. Lu C. In vivo analysis of fibroin heavy chain signal peptide of silkworm Bombyx mori using recombinant baculovirus as vector.Biochem. Biophys. Res. Commun. 2006; 341: 1203-1210Google Scholar Another interesting phenomenon is that the bands of broken fragments of F-POI (GFP) were detected in the NGC, NGCs, and NsGCs groups in western blot (Figure 2C), which may result from the cleavage of GFP during the dissolution process of cocoon silks, as reported previously.2Iizuka T. Sezutsu H. Tatematsu K. Kobayashi I. Yonemura N. Uchino K. Nakajima K. Kojima K. Takabayashi C. Machii H. et al.Colored fluorescent silk made by transgenic silkworms.Adv. Funct. Mater. 2013; 23: 5232-5239Google Scholar,10Long D. Lu W. Zhang Y. Guo Q. Xiang Z. Zhao A. New insight into the mechanism underlying fibroin secretion in silkworm, Bombyx mori.FEBS J. 2015; 282: 89-101Google Scholar,18Long D. Lu W. Zhang Y. Bi L. Xiang Z. Zhao A. An efficient strategy for producing a stable, replaceable, highly efficient transgene expression system in silkworm, Bombyx mori.Sci. Rep. 2015; 5: 8802Google Scholar To extract a mixture of SF and F-POI from cocoons for further material preparation, cocoons should be cut into pieces and degummed to remove the outer sericin layer as described previously.5Saotome T. Hayashi H. Tanaka R. Kinugasa A. Uesugi S. Tatematsu K.-i. Sezutsu H. Kuwabara N. Asakura T. Introduction of VEGF or RGD sequences improves revascularization properties of Bombyx mori silk fibroin produced by transgenic silkworm.J. Mater. Chem. B. 2015; 3: 7109-7116Google Scholar From all four experimental groups exhibiting green fluorescence, only the NG group without a C-terminal domain lost its fluorescence after degumming (Figure 3A). Furthermore, frozen cross-sections of single silk fibers revealed that green fluorescent POI (GFP) was observed in both fibroin and sericin layers in the NG group, while large amounts of F-POI (GFP) were lost after degumming (Figure 3B). Without a complete C-terminal domain, F-POI (GFP) was removed together with soluble sericin during degumming, which resulted in considerable loss of F-POI (GFP) in the extraction process. Consequently, this group was considered unsuitable for the preparation of SF/F-POI hybrid materials.23Rockwood D.N. Preda R.C. Yücel T. Wang X. Lovett M.L. Kaplan D.L. Materials fabrication from Bombyx mori silk fibroin.Nat. Protoc. 2011; 6: 1612-1631Google Scholar Furthermore, larval silk glands were dissected to unravel the specific roles of shortened N- and C-terminal domains in F-POI secretion. As shown in Figure 3C, natural SF is first synthesized by posterior silk gland cells and secreted into the lumen. Then, natural SF is transported into middle silk gland, where sericin is synthesized by middle silk gland cells, secreted into the lumen, and wraps on SF. Finally, core (SF)-shell (sericin)-like silk fibers were extruded through the spinneret of silkworms after packing in anterior silk glands.19Inoue S. Tanaka K. Arisaka F. Kimura S. Ohtomo K. Mizuno S. 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. 2000; 275: 40517-40528Google Scholar We observed that only the posterior silk gland exhibited green fluorescence in the GCs group (Figure 3D). This indicates that F-POI without any N-terminal domain can be synthesized by posterior gland cells, but not secreted into the lumen or subsequently transported into middle silk gland.9Zhao A. Zhao T. Zhang Y. Xia Q. Cheng L. Zhou Z. Xiang Z. Nakagaki M. New and highly efficient expression systems for expressing selectively foreign protein in the silk glands of transgenic silkworm.Transgenic Res. 2010; 19: 29-44Google Scholar In contrast, for the other four groups (NGC, NG, NGCs, and NsGCs) with N-terminal domain or shortened N-terminal domain, green fluorescence was observed throughout the entire gland, indicating that these F-POI (GFP) could accomplish the whole secretion process. These results demonstrate that the shortened N-terminal domain designed in our light-clothing strategy could play a similar function by ensuring the progress of F-POI secretion similar to the N-terminal domain in conventional expression systems. We further prepared frozen cross-sections of silk glands from the four groups with N-terminal domains or shortened N-terminal domains, which were all characterized by complete secretion progress (Figure 3E). F-POI (GFP) in the NG group, i.e., the group without a C-terminal domain, entered the sericin layer in the middle silk gland, and almost all F-POI (GFP) was translocated into the sericin layer in anterior silk glands. After being extruded and spun into cocoons, the majority of F-POI (GFP) still stayed in the sericin layer of silk fibers in the NG group, although some returned to the fibroin layer (Figure 3B). However, the F-POI (GFP) in the other three groups with a C-terminal domain or a shortened C-terminal domain remained in the fibroin layer during the entire secretion process (Figure 3E). Natural SF-h molecules assemble into fibroin units before their secretion into the fibroin layer.19Inoue S. Tanaka K. Arisaka F. Kimura S. Ohtomo K. Mizuno S. 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. 2000; 275: 40517-40528Google Scholar,21Hu W. Lu W. Wei L. Zhang Y. Xia Q. Molecular nature of dominant naked pupa mutation reveals novel insights into silk production in Bombyx mori.Insect Biochem. Mol. Biol. 2019; 109: 52-62Google Scholar We speculate that the retained amino acid residues on the shortened C-terminal domain of F-POI may help F-POI assemble fibroin units with natural SF-h and secrete into the fibroin layer together. F-POI without any C-terminal domain may be in a free state, and can transfer between fibroin and sericin layers during secretion. These results demonstrate that the designed shortened C-terminal domain can prevent the arbitrary transfer of F-POI, similar to the complete C-terminal domain in the conventional expression system. In addition, it should be emphasized that the cocoons of the NG group showed similar fluorescence intensity as that of NGC (Figure 3A), indicating that most of the F-POI could be secreted out without a C-terminal domain. In the above-described sections, we proved that our trimming of the F-POI using the light-clothing strategy could be used to hybridize F-POI, thereby avoiding supernumerary post-translational modifications and ensuring a normal secretion and extraction. Next, the efficacy of our strategy to enhance the activity of functional F-POI was demonstrated by genetic hybridization of a growth factor into SF-based materials. EGF, a potent growth factor widely used for wound healing,24Tada S. Timucin E. Kitajima T. Sezerman O.U. Ito Y. Direct in vitro selection of titanium-binding epidermal growth factor.Biomaterials. 2014; 35: 3497-3503Google Scholar was selected as a functional model POI. A lightly clothed F-POI (EGF) with shortened N- and C-terminal domains (NsECs group) and a heavily clothed F-POI (EGF) with complete N- and C-terminal domains (NEC group) were designed and genetically hybridized into natural SF (Figures S9–S12; see supplemental experimental procedures for more details). Two corresponding transgenic strains were constructed a