The Experts below are selected from a list of 18315 Experts worldwide ranked by ideXlab platform
Donald L Jarvis - One of the best experts on this subject based on the ideXlab platform.
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Silkworms transformed with chimeric Silkworm spider silk genes spin composite silk fibers with improved mechanical properties
Proceedings of the National Academy of Sciences of the United States of America, 2012Co-Authors: Florence Teule, Yun-gen Miao, Malcolm J Fraser, Bonghee Sohn, Joe J Hull, Randolph V Lewis, Donald L JarvisAbstract:The development of a spider silk-manufacturing process is of great interest. However, there are serious problems with natural manufacturing through spider farming, and standard recombinant protein production platforms have provided limited progress due to their inability to assemble spider silk proteins into fibers. Thus, we used piggyBac vectors to create transgenic Silkworms encoding chimeric Silkworm/spider silk proteins. The silk fibers produced by these animals were composite materials that included chimeric Silkworm/spider silk proteins integrated in an extremely stable manner. Furthermore, these composite fibers were, on average, tougher than the parental Silkworm silk fibers and as tough as native dragline spider silk fibers. These results demonstrate that Silkworms can be engineered to manufacture composite silk fibers containing stably integrated spider silk protein sequences, which significantly improve the overall mechanical properties of the parental Silkworm silk fibers.
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Silkworms transformed with chimeric Silkworm/spider silk genes spin composite silk fibers with improved mechanical properties
Proceedings of the National Academy of Sciences of the United States of America, 2012Co-Authors: Florence Teule, Yun-gen Miao, Malcolm J Fraser, Bonghee Sohn, Joe J Hull, Randolph V Lewis, Young-soo Kim, Donald L JarvisAbstract:The development of a spider silk-manufacturing process is of great interest. However, there are serious problems with natural manufacturing through spider farming, and standard recombinant protein production platforms have provided limited progress due to their inability to assemble spider silk proteins into fibers. Thus, we used piggyBac vectors to create transgenic Silkworms encoding chimeric Silkworm/spider silk proteins. The silk fibers produced by these animals were composite materials that included chimeric Silkworm/spider silk proteins integrated in an extremely stable manner. Furthermore, these composite fibers were, on average, tougher than the parental Silkworm silk fibers and as tough as native dragline spider silk fibers. These results demonstrate that Silkworms can be engineered to manufacture composite silk fibers containing stably integrated spider silk protein sequences, which significantly improve the overall mechanical properties of the parental Silkworm silk fibers.
Boxiong Zhong - One of the best experts on this subject based on the ideXlab platform.
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Recombinant Silk Proteins with Additional Polyalanine Have Excellent Mechanical Properties.
International journal of molecular sciences, 2021Co-Authors: Shuo Zhao, Jinghua Ruan, Xiaoxiao Wang, Xiaoli Tang, Boxiong ZhongAbstract:This paper explores the structures of exogenous protein molecules that can effectively improve the mechanical properties of Silkworm silk. Several transgenic vectors fused with the Silkworm fibroin light chain and type 3 repeats in different multiples of the ampullate dragline silk protein 1 (MaSp1) from black widow spider with different lengths of the polyalanine motifs were constructed for this study. Transgenic Silkworms were successfully obtained by piggyBac-mediated microinjection. Molecular detection showed that foreign proteins were successfully secreted and contained within the cocoon shells. According to the prediction of PONDR® VSL2 and PONDR® VL-XT, the type 3 repeats and the polyalanine motif of the MaSp1 protein were amorphous. The results of FTIR analysis showed that the content of β-sheets in the silk of transgenic Silkworms engineered with transgenic vectors with additional polyalanine was significantly higher than that of wild-type Silkworm silk. Additionally, silk with a higher β-sheet content had better fracture strength and Young’s modulus. The mechanical properties of silk with longer chains of exogenous proteins were improved. In general, our results provide theoretical guidance and technical support for the large-scale production of excellent bionic silk.
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High-efficiency production of human serum albumin in the posterior silk glands of transgenic Silkworms, Bombyx mori L
2018Co-Authors: Qiujie Qian, Zhengying You, Jiaqian Che, Yiran Wang, Shaohua Wang, Boxiong ZhongAbstract:Human serum albumin (HSA) is an important biological preparation with a variety of biological functions in clinical applications. In this study, the mRNA of a fusion transposase derived from the pESNT-PBase plasmid and a pBHSA plasmid containing the HSA gene under the control of a fibroin light chain (FL) promoter were co-injected into fertilized eggs. Fifty-six transgenic Silkworm pedigrees expressing theexogenous recombinant HSA (rHSA) in the posterior silk glands (PSGs) with stable inheritance were successfully obtained. The SDS-PAGE and Western blot results confirmed that the rHSA was secreted into the transgenic Silkworm cocoon, and the rHSA could be easily extracted with phosphate-buffered saline (PBS). In our research, the isolated highest amount rHSA constituted up to 29.1% of the total soluble protein of the cocoon shell, indicating that the transgenic Silkworm produced an average of 17.4 μg/mg of rHSA in the cocoon shell. The production of soluble rHSA in the PSGs by means of generating transgenic Silkworms is a novel approach, whereby a large amount of virus-free and functional HSA can be produced through the simple rearing of Silkworms.
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Comparative Proteomic Analysis of Posterior Silk Glands of Wild and Domesticated Silkworms Reveals Functional Evolution during Domestication
Journal of Proteome Research, 2017Co-Authors: Jianying Li, Jianshe Liang, Jian-ke Li, Zhen-dong Jiang, Xiaogang Ye, Meiyu Wu, Dan Zhao, Boxiong ZhongAbstract:The wild Silkworm Bombyx mandarina was domesticated to produce silk in China approximately 5000 years ago. Silk production is greatly improved in the domesticated Silkworm B. mori, but the molecular basis of the functional evolution of silk gland remains elusive. We performed shotgun proteomics with label-free quantification analysis and identified 1012 and 822 proteins from the posterior silk glands (PSGs) of wild Silkworms on the third and fifth days of the fifth instar, respectively, with 128 of these differentially expressed. Bioinformatics analysis revealed that, with the development of the PSG, the up-regulated proteins were mainly involved in the ribosome pathway, similar to what we previously reported for B. mori. Additionally, we screened 50 proteins with differential expression between wild and domesticated Silkworms that might be involved in domestication at the two stages. Interestingly, the up-regulated proteins in domesticated compared to wild Silkworms were enriched in the ribosome pathway,...
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Comparative Proteomic Analysis of Posterior Silk Glands of Wild and Domesticated Silkworms Reveals Functional Evolution during Domestication
2017Co-Authors: Fang Cai, Jianshe Liang, Zhen-dong Jiang, Dan Zhao, Zhengying You, Boxiong ZhongAbstract:The wild Silkworm Bombyx mandarina was domesticated to produce silk in China approximately 5000 years ago. Silk production is greatly improved in the domesticated Silkworm B. mori, but the molecular basis of the functional evolution of silk gland remains elusive. We performed shotgun proteomics with label-free quantification analysis and identified 1012 and 822 proteins from the posterior silk glands (PSGs) of wild Silkworms on the third and fifth days of the fifth instar, respectively, with 128 of these differentially expressed. Bioinformatics analysis revealed that, with the development of the PSG, the up-regulated proteins were mainly involved in the ribosome pathway, similar to what we previously reported for B. mori. Additionally, we screened 50 proteins with differential expression between wild and domesticated Silkworms that might be involved in domestication at the two stages. Interestingly, the up-regulated proteins in domesticated compared to wild Silkworms were enriched in the ribosome pathway, which is closely related to cell size and translation capacity. Together, these results suggest that functional evolution of the PSG during domestication was driven by reinforcing the advantageous pathways to increase the synthesis efficiency of silk proteins in each cell and thereby improve silk yield
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Transgenic breeding of anti‐Bombyx mori L. nuclear polyhedrosis virus Silkworm Bombyx mori
Acta biochimica et biophysica Sinica, 2008Co-Authors: Hui-juan Yang, Wei Fan, Hao Wei, Jin-wei Zhang, Zhong-hua Zhou, Jianrong Lin, Nong Ding, Boxiong ZhongAbstract:Silkworm strains resistant to Bombyx mori L. nuclear polyhedrosis virus were obtained through transgenic experiments. piggyBac transposon with an A3 promoter were randomly inserted into the Silkworm, driving the enhanced green fluorescent protein (EGFP) reporter gene into the Silkworm genome. Polymerase chain reaction results verified the insertion of the extraneous EGFP gene, and fluorescence microscopy showed that the EGFP was expressed in the midgut tissue. The morbidity ratio of the nuclear polyhedrosis decreased from 90% in the original Silkworm strain to 66.7% in the transgenic Silkworm strain. Compared with the resistance to the Bombyx mori L. nuclear polyhedrosis virus in the Qiufeng strain, which is commonly used in the production, there was an increase of 33 centesimal points in the transgenic Silkworms. The antivirotic character in the Chunhua×Qiuyue strain, which was bred from a different transgenic family, was about 10 centesimal points higher than that in the Qiufeng×Baiyu, another crossbreed used in production. Our results indicated a good application value of the transposon-inserted mutation in the breeding of antiBmNPV Silkworm strain.
Enoch Y Park - One of the best experts on this subject based on the ideXlab platform.
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Alteration of a recombinant protein N-glycan structure in Silkworms by partial suppression of N-acetylglucosaminidase gene expression
Biotechnology Letters, 2017Co-Authors: Tatsuya Kato, Kotaro Kikuta, Ayumi Kanematsu, Sachiko Kondo, Hirokazu Yagi, Koichi Kato, Enoch Y ParkAbstract:Objective To synthesize complex type N -glycans in Silkworms, shRNAs against the fused lobe from Bombyx mori (BmFDL), which codes N -acetylglucosaminidase (GlcNAcase) in the Golgi, was expressed by recombinant B. mori nucleopolyhedrovirus (BmNPV) in Silkworm larvae. Results Expression was under the control of the actin promoter of B. mori or the U6-2 and i.e.-2 promoters from Orgyia pseudotsugata multiple nucleopolyhedrovirus (OpMNPV). The reduction of specific GlcNAcase activity was observed in Bm5 cells and Silkworm larvae using the U6-2 promoter. In Silkworm larvae, the partial suppression of BmFDL gene expression was observed. When shRNA against BmFDL was expressed under the control of U6-2 promoter, the Man_3GlcNAc(Fuc)GlcNAc structure appeared in a main N -glycans of recombinant human IgG. These results suggested that the control of BmFDL expression by its shRNA in Silkworms caused the modification of its N -glycan synthetic pathway, which may lead to the alteration of N -glycans in the expressed recombinant proteins. Conclusions Suppression of BmFDL gene expression by shRNA is not sufficient to synthesize complex N -glycans in Silkworm larvae but can modify the N -glycan synthetic pathway.
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Advanced Protein Expression Using Bombyx mori Nucleopolyhedrovirus (BmNPV) Bacmid in Silkworm
Short Views on Insect Genomics and Proteomics, 2015Co-Authors: Tatsuya Kato, Enoch Y ParkAbstract:An expression system using a Bombyx mori nucleopolyhedrovirus (BmNPV) bacmid has been developed and used for efficient expression of protein using Silkworm. Silkworm can sustain large-scale production of recombinant proteins due to its ease of rearing and scaling-up. Our chapter focuses on the modification of a BmNPV bacmid for a more efficient protein expression system. For example, we discuss how to achieve construction of a stronger promoter, less proteolytic degradation of expressed proteins, and a chaperone-coexpressed expression system. We describe the application of functional BmNPV particles purified from Silkworm hemolymph to vaccines, antibody production, and transmembrane protein analysis. For human use, the major problem of proteins produced in Silkworm is contamination by adventitious agents and protein quality. Of special concern is that N-glycosylation in Silkworms is of a high-mannose type in most cases, which is different from the complex type found in mammals. We end by looking to future prospects for integration/applications of protein expression systems with Silkworm biotechnology.
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Silkworm expression system as a platform technology in life science
Applied Microbiology and Biotechnology, 2010Co-Authors: Tatsuya Kato, Mizuho Kajikawa, Katsumi Maenaka, Enoch Y ParkAbstract:Many recombinant proteins have been successfully produced in Silkworm larvae or pupae and used for academic and industrial purposes. Several recombinant proteins produced by Silkworms have already been commercialized. However, construction of a recombinant baculovirus containing a gene of interest requires tedious and troublesome steps and takes a long time (3–6 months). The recent development of a bacmid, Escherichia coli and Bombyx mori shuttle vector, has eliminated the conventional tedious procedures required to identify and isolate recombinant viruses. Several technical improvements, including a cysteine protease or chitinase deletion bacmid and chaperone-assisted expression and coexpression, have led to significantly increased protein yields and reduced costs for large-scale production. Terminal N -acetyl glucosamine and galactose residues were found in the N -glycan structures produced by Silkworms, which are different from those generated by insect cells. Genomic elucidation of Silkworm has opened a new chapter in utilization of Silkworm. Transgenic Silkworm technology provides a stable production of recombinant protein. Baculovirus surface display expression is one of the low-cost approaches toward Silkworm larvae-derived recombinant subunit vaccines. The expression of pharmaceutically relevant proteins, including cell/viral surface proteins, membrane proteins, and guanine nucleotide-binding protein (G protein) coupled receptors, using Silkworm larvae or cocoons has become very attractive. Silkworm biotechnology is an innovative and easy approach to achieve high protein expression levels and is a very promising platform technology in the field of life science. Like the “Silkroad,” we expect that the “Bioroad” from Asia to Europe will be established by the Silkworm expression system.
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Construction of a cysteine protease deficient Bombyx mori multiple nucleopolyhedrovirus bacmid and its application to improve expression of a fusion protein.
Journal of virological methods, 2007Co-Authors: Masato Hiyoshi, Tatsuya Kato, Ayano Kageshima, Enoch Y ParkAbstract:The bacmid system of BmMNPV with cysteine protease gene deletion (CPD-BmMNPV bacmid) was constructed using the lambda recombination system. The protease activities of Bombyx mori cells and Silkworm larvae infected with this CPD-BmMNPV bacmid were reduced by 94% and 85%, respectively. By using this system, a GFP(uv)-beta1,3-N-acetylglucosaminyltransferase 2 (GFP(uv)-beta3GnT2) fusion protein was successfully expressed in Silkworm larvae with less protein degradation and without larvae liquefaction; beta3GnT activity improved 30%. This CPD-BmMNPV bacmid system provides rapid protein production in Silkworms and can be used for the production of recombinant eukaryotic proteins without proteolytic degradation.
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Construction of a cysteine protease deficient Bombyx mori multiple nucleopolyhedrovirus bacmid and its application to improve expression of a fusion protein.
Journal of Virological Methods, 2007Co-Authors: Masato Hiyoshi, Tatsuya Kato, Ayano Kageshima, Enoch Y ParkAbstract:Abstract The bacmid system of BmMNPV with cysteine protease gene deletion (CPD-BmMNPV bacmid) was constructed using the lambda recombination system. The protease activities of Bombyx mori cells and Silkworm larvae infected with this CPD-BmMNPV bacmid were reduced by 94% and 85%, respectively. By using this system, a GFPuv-β1,3-N-acetylglucosaminyltransferase 2 (GFPuv-β3GnT2) fusion protein was successfully expressed in Silkworm larvae with less protein degradation and without larvae liquefaction; β3GnT activity improved 30%. This CPD-BmMNPV bacmid system provides rapid protein production in Silkworms and can be used for the production of recombinant eukaryotic proteins without proteolytic degradation.
Yun-gen Miao - One of the best experts on this subject based on the ideXlab platform.
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Silkworms transformed with chimeric Silkworm spider silk genes spin composite silk fibers with improved mechanical properties
Proceedings of the National Academy of Sciences of the United States of America, 2012Co-Authors: Florence Teule, Yun-gen Miao, Malcolm J Fraser, Bonghee Sohn, Joe J Hull, Randolph V Lewis, Donald L JarvisAbstract:The development of a spider silk-manufacturing process is of great interest. However, there are serious problems with natural manufacturing through spider farming, and standard recombinant protein production platforms have provided limited progress due to their inability to assemble spider silk proteins into fibers. Thus, we used piggyBac vectors to create transgenic Silkworms encoding chimeric Silkworm/spider silk proteins. The silk fibers produced by these animals were composite materials that included chimeric Silkworm/spider silk proteins integrated in an extremely stable manner. Furthermore, these composite fibers were, on average, tougher than the parental Silkworm silk fibers and as tough as native dragline spider silk fibers. These results demonstrate that Silkworms can be engineered to manufacture composite silk fibers containing stably integrated spider silk protein sequences, which significantly improve the overall mechanical properties of the parental Silkworm silk fibers.
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Silkworms transformed with chimeric Silkworm/spider silk genes spin composite silk fibers with improved mechanical properties
Proceedings of the National Academy of Sciences of the United States of America, 2012Co-Authors: Florence Teule, Yun-gen Miao, Malcolm J Fraser, Bonghee Sohn, Joe J Hull, Randolph V Lewis, Young-soo Kim, Donald L JarvisAbstract:The development of a spider silk-manufacturing process is of great interest. However, there are serious problems with natural manufacturing through spider farming, and standard recombinant protein production platforms have provided limited progress due to their inability to assemble spider silk proteins into fibers. Thus, we used piggyBac vectors to create transgenic Silkworms encoding chimeric Silkworm/spider silk proteins. The silk fibers produced by these animals were composite materials that included chimeric Silkworm/spider silk proteins integrated in an extremely stable manner. Furthermore, these composite fibers were, on average, tougher than the parental Silkworm silk fibers and as tough as native dragline spider silk fibers. These results demonstrate that Silkworms can be engineered to manufacture composite silk fibers containing stably integrated spider silk protein sequences, which significantly improve the overall mechanical properties of the parental Silkworm silk fibers.
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cloning and expression of a cellulase gene in the Silkworm bombyx mori by improved bac to bac bmnpv baculovirus expression system
Molecular Biology Reports, 2010Co-Authors: Xinghua Li, Roy Bhaskar, Jiabiao Hu, Huajun Yang, Fang Zhou, Dan Wang, Yun-gen MiaoAbstract:Cellulases catalyze the hydrolysis of cellulose which are mainly three types: endoglucanases, cellobiohydrolases and β-glucosidases. It can be used in converting cellulosic biomass to glucose that can be used in different applications such as production of fuel ethanol, animal feed, waste water treatment and in brewing industry. In this paper, we cloned a 1380-bp endoglucanase I (EG I) gene from mycelium of filamentous fungus Trichoderma viride strain AS 3.3711 using PCR-based exon splicing methods, and expressed the recombinant EG I mature peptide protein in both Silkworm BmN cell line and Silkworm larvae with a newly established Bac-to-Bac/BmNPV mutant baculovirus expression system, which lacks the virus-encoded chitinase (chiA) and cathepsin (v-cath) genes of Bombyx mori nucleopolyhedrovirus (BmNPV). An around 49-kDa protein was visualized after mBacmid/BmNPV/EG I infection, and the maximum expression in Silkworm larvae was at 84 h post-infection. The ANOVA showed that the enzymes from recombinant baculoviruses infected Silkworms exhibited significant maximum enzyme activity at the environmental condition of pH 7.0 and temperature 50°C. It was stable at pH range from 5.0 to 10.0 and at temperature range from 50 to 60°C, and increased 24.71 and 22.84% compared with that from wild baculoviruses infected Silkworms and normal Silkworms, respectively. The availability of large quantities of EG I that the Silkworm provides maybe greatly facilitate the future research and the potential application in industries.
Florence Teule - One of the best experts on this subject based on the ideXlab platform.
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Silkworms transformed with chimeric Silkworm spider silk genes spin composite silk fibers with improved mechanical properties
Proceedings of the National Academy of Sciences of the United States of America, 2012Co-Authors: Florence Teule, Yun-gen Miao, Malcolm J Fraser, Bonghee Sohn, Joe J Hull, Randolph V Lewis, Donald L JarvisAbstract:The development of a spider silk-manufacturing process is of great interest. However, there are serious problems with natural manufacturing through spider farming, and standard recombinant protein production platforms have provided limited progress due to their inability to assemble spider silk proteins into fibers. Thus, we used piggyBac vectors to create transgenic Silkworms encoding chimeric Silkworm/spider silk proteins. The silk fibers produced by these animals were composite materials that included chimeric Silkworm/spider silk proteins integrated in an extremely stable manner. Furthermore, these composite fibers were, on average, tougher than the parental Silkworm silk fibers and as tough as native dragline spider silk fibers. These results demonstrate that Silkworms can be engineered to manufacture composite silk fibers containing stably integrated spider silk protein sequences, which significantly improve the overall mechanical properties of the parental Silkworm silk fibers.
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Silkworms transformed with chimeric Silkworm/spider silk genes spin composite silk fibers with improved mechanical properties
Proceedings of the National Academy of Sciences of the United States of America, 2012Co-Authors: Florence Teule, Yun-gen Miao, Malcolm J Fraser, Bonghee Sohn, Joe J Hull, Randolph V Lewis, Young-soo Kim, Donald L JarvisAbstract:The development of a spider silk-manufacturing process is of great interest. However, there are serious problems with natural manufacturing through spider farming, and standard recombinant protein production platforms have provided limited progress due to their inability to assemble spider silk proteins into fibers. Thus, we used piggyBac vectors to create transgenic Silkworms encoding chimeric Silkworm/spider silk proteins. The silk fibers produced by these animals were composite materials that included chimeric Silkworm/spider silk proteins integrated in an extremely stable manner. Furthermore, these composite fibers were, on average, tougher than the parental Silkworm silk fibers and as tough as native dragline spider silk fibers. These results demonstrate that Silkworms can be engineered to manufacture composite silk fibers containing stably integrated spider silk protein sequences, which significantly improve the overall mechanical properties of the parental Silkworm silk fibers.
