The Experts below are selected from a list of 321 Experts worldwide ranked by ideXlab platform
David L Kaplan - One of the best experts on this subject based on the ideXlab platform.
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construction cloning and expression of Synthetic Genes encoding spider dragline silk
Biochemistry, 1995Co-Authors: John T Prince, Kevin P Mcgrath, Carla M Digirolamo, David L KaplanAbstract:: Synthetic Genes encoding recombinant spider silk proteins have been constructed, cloned, and expressed. Protein sequences were derived from Nephila clavipes dragline silk proteins and reverse-translated to the corresponding DNA sequences. Codon selection was chosen to maximize expression levels in Escherichia coli. DNA "monomer" sequences were multimerized to encode high molecular weight Synthetic spider silks using a "head-to-tail" construction strategy. Multimers were cloned into a prokaryotic expression vector and the encoded silk proteins were expressed in E. coli upon induction with IPTG. Four multimer, ranging in size from 14.7 to 41.3 kDa, were chosen for detailed analysis. These proteins were isolated by immobilized metal affinity chromatography and purified using reverse-phase HPLC. The composition and identity of the purified proteins were confirmed by amino acid composition analysis, N-terminal sequencing, laser desorption mass spectroscopy, and Western analysis using antibodies reactive to native spider dragline silk. Circular dichroism measurements indicate that the Synthetic spider silks have substantial beta-sheet structure.
L A Bedzyk - One of the best experts on this subject based on the ideXlab platform.
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production of Synthetic spider dragline silk protein in pichia pastoris
Applied Microbiology and Biotechnology, 1997Co-Authors: S R Fahnestock, L A BedzykAbstract:The methylotrophic yeast Pichia pastoris was tested as a host for the production of long, repetitive protein polymers. Synthetic Genes for a designed analog of a spider dragline silk protein were readily expressed at high levels under control of the methanol-inducible AOX1 promoter. Transformants containing multiple gene copies produced elevated levels of silk protein, but of a variety of altered sizes as a result of gene rearrangements at the time of transformation. Genes up to 3000 codons in length or longer could be expressed with no evidence of the prevalent truncated synthesis observed for similar Genes in Escherichia coli, though Genes longer than 1600 codons were expressed less efficiently than shorter Genes. Silk-producing P. pastoris strains were stable without selection for at least 100 doublings.
Hitoshi Shimano - One of the best experts on this subject based on the ideXlab platform.
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sterol regulatory element binding protein family as global regulators of lipid Synthetic Genes in energy metabolism
Vitamins and Hormones Series, 2002Co-Authors: Hitoshi ShimanoAbstract:Sterol regulatory element-binding proteins (SREBPs) have been established as lipid Synthetic transcription factors for cholesterol and fatty acid synthesis. SREBPs are synthesized as membrane-bound precursors with their N-terminal active portions entering the nucleus to activate target Genes after proteolytic cleavage in a sterol-regulated manner. This cleavage step is regulated by a putative sterol-sensing molecule, SREBP-activating protein (SCAP), that forms a complex with SREBPs and traffics between the rough endoplasmic reticulum and Golgi. DNA cis-elements that SREBPs bind, originally identified as sterol-regulatory elements (SREs), now expands to a variety of SRE-like sequences and some of E-boxes, which makes SREBPs eligible to regulate a wide range of lipid Genes. Animal experiments including transgenic and knockout mice suggest that three isoforms, SREBP-1a, -1c, and -2, have different roles in lipid synthesis. In differentiated tissues and organs, SREBP-1c is involved in fatty acid, whereas SREBP-2 plays a major role in regulation of cholesterol synthesis. SREBP-1a is expressed in growing cells, providing both cholesterol and fatty acids that are required for membrane synthesis. SREBP-1c seems to be a mediator for insulin/glucose signaling to lipoGenesis, and could be involved in insulin resistance, remnant lipoproteins, and fatty livers. Future studies in this field will certainly focus on understanding the molecular mechanisms sensing cellular sterol and energy states leading to the activation of SREBP-mediated gene transcription.
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sterol regulatory element binding proteins srebps transcriptional regulators of lipid Synthetic Genes
Progress in Lipid Research, 2001Co-Authors: Hitoshi ShimanoAbstract:Roles of sterol regulatory element-binding proteins (SREBPs) have been established as lipid Synthetic transcription factors especially for cholesterol and fatty acid synthesis. SREBPs have unique characteristics. Firstly, they are membrane-bound proteins and the N-terminal active portions enter nucleus to activate their target Genes after proteolytic cleavage, which requires sterol-sensing molecule, SREBP-activating protein (SCAP) and is crucial for sterol-regulation. Secondly, they bind and activate sterol-regulatory (SREs) containing promoters as well as some E-boxes, which makes SREBPs eligible to regulate a wide range of lipid Genes. Finally, three isoforms, SREBP-1a-1c, and have different roles in lipid synthesis. In vivo studies using transgenic and knockout mice suggest that SREBP-1 seems to be involved in energy metabolism including fatty acid and glucose/insulin metabolism, whereas SREBP-2 is specific to cholesterol synthesis. Future studies will be focused on understanding molecular mechanisms sensing cellular sterol and energy states where SREBPs are deeply involved.
Thomas Scheibel - One of the best experts on this subject based on the ideXlab platform.
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recombinant production of spider silk proteins
Advances in Applied Microbiology, 2013Co-Authors: Aniela Heidebrecht, Thomas ScheibelAbstract:Abstract Natural spider silk fibers combine extraordinary properties such as stability and flexibility which results in a toughness superseding that of all other fiber materials. As the spider’s aggressive territorial behavior renders their farming not feasible, the biotechnological production of spider silk proteins (spidroins) is essential in order to investigate and employ them for applications. In order to accomplish this task, two approaches have been tested: firstly, the expression of partial cDNAs, and secondly, the expression of Synthetic Genes in several host organisms, including bacteria, yeast, plants, insect cells, mammalian cells, and transgenic animals. The experienced problems include genetic instability, limitations of the translational and transcriptional machinery, and low solubility of the produced proteins. Here, an overview of attempts to recombinantly produce spidroins will be given, and advantages and disadvantages of the different approaches and host organisms will be discussed.
John T Prince - One of the best experts on this subject based on the ideXlab platform.
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construction cloning and expression of Synthetic Genes encoding spider dragline silk
Biochemistry, 1995Co-Authors: John T Prince, Kevin P Mcgrath, Carla M Digirolamo, David L KaplanAbstract:: Synthetic Genes encoding recombinant spider silk proteins have been constructed, cloned, and expressed. Protein sequences were derived from Nephila clavipes dragline silk proteins and reverse-translated to the corresponding DNA sequences. Codon selection was chosen to maximize expression levels in Escherichia coli. DNA "monomer" sequences were multimerized to encode high molecular weight Synthetic spider silks using a "head-to-tail" construction strategy. Multimers were cloned into a prokaryotic expression vector and the encoded silk proteins were expressed in E. coli upon induction with IPTG. Four multimer, ranging in size from 14.7 to 41.3 kDa, were chosen for detailed analysis. These proteins were isolated by immobilized metal affinity chromatography and purified using reverse-phase HPLC. The composition and identity of the purified proteins were confirmed by amino acid composition analysis, N-terminal sequencing, laser desorption mass spectroscopy, and Western analysis using antibodies reactive to native spider dragline silk. Circular dichroism measurements indicate that the Synthetic spider silks have substantial beta-sheet structure.
