The formation of a quaternary structure by recombinant analogs of spider silk proteins

Sokolova, Olga S.; Богуш, В. Г.; Давыдова, Л. И.; Polevova, Svetlana; Antonov, S. A.; Neretina, Tatyana V.; Klinov, Dmitry V.; Дебабов, В. Г.; Kirpichnikov, M. P.

doi:10.1134/s0026893310010188

Cited by 8 publications

(8 citation statements)

References 21 publications

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“…ProteinMolecular weight (kDa)bTargetingCultureYield (g/Lc)Reference Silk-like proteins Nephila clavipes MaSp1 mimics64/127dIS/B0.2–1Fahnestock and Bedzyk, 1997 N. clavipes MaSp1 mimics64/127Een/s≤6 μg/OD 600 Fahnestock et al, 2000 N. clavipes MaSp1 mimics ± COL~15–30EeSn/sTeulé et al, 2003 Nephila sp. MaSp1 and MaSp2 mimics94/113IBn/sBogush et al, 2009; Sokolova et al, 2010 N. clavipes MaSp1 and MaSp2 mimics + NTD + CTD44EBn/sGaines and Marcotte Jr., 2011 Euprosthenops australis MaSp1 mimic + CTD32EeSn/sJansson et al, 2016Silk-inspired octapeptide repeats28–32EB0.9–2.9fWerten et al, 2008 Gelatins (nonhydroxylated) Murine α1(I) and α1(III) fragments21–53EB2–15fWerten et al, 1999, Werten and de Wolf, 2005Artificial hydrophilic gelatin C P 4 37EB3–6fWerten et al, 2001Human α1(I), α1(II), and α1(III) fragments5–90EBn/sOlsen et al, 2003Human α1(I) fragment9EB1.5fOlsen et al, 2005…”

Section: Protein Polymers Produced In P Pastorismentioning

confidence: 99%

Production of protein-based polymers in Pichia pastoris

Werten

Eggink

Stuart

et al. 2019

Biotechnology Advances

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Materials science and genetic engineering have joined forces over the last three decades in the development of so-called protein-based polymers. These are proteins, typically with repetitive amino acid sequences, that have such physical properties that they can be used as functional materials. Well-known natural examples are collagen, silk, and elastin, but also artificial sequences have been devised. These proteins can be produced in a suitable host via recombinant DNA technology, and it is this inherent control over monomer sequence and molecular size that renders this class of polymers of particular interest to the fields of nanomaterials and biomedical research. Traditionally, Escherichia coli has been the main workhorse for the production of these polymers, but the methylotrophic yeast Pichia pastoris is finding increased use in view of the often high yields and potential bioprocessing benefits. We here provide an overview of protein-based polymers produced in P. pastoris . We summarize their physicochemical properties, briefly note possible applications, and detail their biosynthesis. Some challenges that may be faced when using P. pastoris for polymer production are identified: (i) low yields and poor process control in shake flask cultures; i.e. , the need for bioreactors, (ii) proteolytic degradation, and (iii) self-assembly in vivo . Strategies to overcome these challenges are discussed, which we anticipate will be of interest also to readers involved in protein expression in P. pastoris in general.

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Section: Protein Polymers Produced In P Pastorismentioning

confidence: 99%

Production of protein-based polymers in Pichia pastoris

Werten

Eggink

Stuart

et al. 2019

Biotechnology Advances

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“…Due to the possibility of high‐level protein production, the yeast P. pastoris has been frequently used as host for production of a wide range of recombinant proteins, including silk‐derived and silk‐like proteins . The advantage of using the secretory pathway in P. pastoris has also been utilized to secrete produced silk proteins into the surrounding growth medium.…”

Section: Discussionmentioning

confidence: 99%

“…Regardless of using secretion or not, produced and purified silk proteins from P. pastoris have so far not been able to mimic the inherent property of natural silk proteins to self‐assemble into silk–like fibrous materials under physiological conditions. However, dissolution of obtained silk proteins in organic solvents, followed by treatment with ethanol or methanol, has promoted assembly into fibril‐like structures and allowed fiber assembly via electrospinning , as well as artificial spinning through a microspinneret .…”

Section: Discussionmentioning

confidence: 99%

“…In addition, two synthetic silk protein monomers, 1F9 and 2E12, derived from the spiders Nephila clavipes and Nephila madagascariensis , respectively have been intracellularly produced in P. pastoris under control of a methanol‐inducible promoter. After dissolving freeze‐dried protein in lithium chloride and formic acid, centimeter‐long fibers could be formed upon extrusion of the protein solution through a microspinneret into an ethanol bath .…”

Section: Introductionmentioning

confidence: 99%

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Functionalized silk assembled from a recombinant spider silk fusion protein (Z‐4RepCT) produced in the methylotrophic yeast Pichia pastoris

Jansson

Lau

Ishida

et al. 2016

Biotechnology Journal

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Functional biological materials are a growing research area with potential applicability in medicine and biotechnology. Using genetic engineering, the possibility to introduce additional functions into spider silk-based materials has been realized. Recently, a recombinant spider silk fusion protein, Z-4RepCT, was produced intracellularly in Escherichia coli and could after purification self-assemble into silk-like fibers with ability to bind antibodies via the IgG-binding Z domain. In this study, the use of the methylotrophic yeast Pichia pastoris for production of Z-4RepCT has been investigated. Temperature, pH and production time were influencing the amount of soluble Z-4RepCT retrieved from the extracellular fraction. Purification of secreted Z-4RepCT resulted in a mixture of full-length and degraded silk proteins that failed to self-assemble into fibers. A position in the C-terminal domain of 4RepCT was identified as being subjected to proteolytic cleavage by proteases in the Pichia culture supernatant. Moreover, the C-terminal domain was subjected to glycosylation during production in P. pastoris. These observed alterations of the CT domain are suggested to contribute to the failure in fiber assembly. As alternative approach, Z-4RepCT retrieved from the intracellular fraction, which was less degraded, was used and shown to retain ability to assemble into silk-like fibers after enzymatic deglycosylation.

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“…Разница в степени нанопористости также может быть связана с частичным гидролизом фиброина в процессе растворения его в муравьиной кислоте c LiCl и влиянием продуктов гидролиза на формирование матрикса. Некоторые исследования показывают, что муравьиная кислота может оказывать существенное влияние на молекулярную массу фиброина шелка [18], в то время как белок рекомбинантного спидроина rS1/9 остается стабильным в кислотной среде [19]. Однако эта гипотеза требует дальнейших исследований.…”

Section: результаты и обсуждениеunclassified

Comparative Analysis of Three-Dimensional Nanostructure of Porous Biocompatible Scaffolds Made of Recombinant Spidroin and Silk Fibroin for Regenerative Medicine

Agapova

Ефимов

Moisenovich

et al. 2015

RJTAO

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Aim.To perform a comparison of three-dimensional nanostructure of porous biocompatible scaffolds made of fibroinBombix moriand recombinant spidroin rS1/9.Materials and methods.Three-dimensional porous scaffolds were produced by salt leaching technique. The comparison of biological characteristics of the scaffolds shows that adhesion and proliferation of mouse fibroblastsin vitroon these two types of scaffolds do not differ significantly. Comparative experimentsin vivoshow that regeneration of bone tissue of rats is faster with implantation of recombinant spidroin scaffolds. Three-dimensional nanostructure of scaffolds and interconnectivity of nanopores were studied with scanning probe nanotomography (SPNT) to explain higher regenerative activity of spidroin-based scaffolds.Results.Significant differences were detected in the integral density and volume of pores: the integral density of nanopores detected on 2D AFM images is 46 μm–2 and calculated volume porosity is 24% in rS1/9-based scaffolds; in fibroin-based three-dimensional structures density of nanopores and calculated volume porosity were 2.4 μm–2 and 0.5%, respectively. Three-dimensional reconstruction system of nanopores and clusters of interconnected nanopores in rS1/9-based scaffolds showed that volume fraction of pores interconnected in percolation clusters is 35.3% of the total pore volume or 8.4% of the total scaffold volume.Conclusion.Scanning probe nanotomography method allows obtaining unique information about topology of micro – and nanopore systems of artificial biostructures. High regenerative activity of rS1/9-based scaffolds can be explained by higher nanoporosity of the scaffolds.

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The formation of a quaternary structure by recombinant analogs of spider silk proteins

Cited by 8 publications

References 21 publications

Production of protein-based polymers in Pichia pastoris

Production of protein-based polymers in Pichia pastoris

Functionalized silk assembled from a recombinant spider silk fusion protein (Z‐4RepCT) produced in the methylotrophic yeast Pichia pastoris

Comparative Analysis of Three-Dimensional Nanostructure of Porous Biocompatible Scaffolds Made of Recombinant Spidroin and Silk Fibroin for Regenerative Medicine

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