Size and Density of Protein Inclusion Bodies

Taylor, G.L.; Hoare, M.; Gray, DJ; Marston, Fiona A. O.

doi:10.1038/nbt0686-553

Cited by 115 publications

(65 citation statements)

References 9 publications

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“…Einstein (Investigations on the theory of Brownian movement, 1926) obtained the following theoretical relation for the viscosity of identical non-interacting rigid spherical particle suspensions at low particle volume fractions: Whole cells bacterial 0.5-5.0 (Agerkvist and Enfors, 1990;Bowden, 1985;Harrison, 1991;Hayashi et al, 2001b;Kula et al, 1990) 1,080-1,120 (Erbeldinger et al, 1998;Wong et al, 1997b) 3.0-5.0 (Hayashi et al, 2001a,b Bowden, 1985;Harrison, 1991;Kula et al, 1990;Siddiqi et al, 1996) $1,040 (Lipschutz et al, 2000;Nikolai and Hu, 1992) - Agerkvist and Enfors, 1990;Bowden, 1985;Kula et al, 1990;Wong et al, 1997a;Wong et al, 1997b) 1,061-1,090 (Wong et al, 1997b) $2.8 (Wangsa-Wirawan et al, 2001b)* Yeast 0.05-8.0 (Kula et al, 1990;Siddiqi et al, 1996) --Inclusion bodies 0.05-1.2 (Taylor et al, 1986;…”

Section: Suspension Characteristicsmentioning

confidence: 99%

Strategy for selection of methods for separation of bioparticles from particle mixtures

Hee

Hoeben

Lans

et al. 2006

Biotech & Bioengineering

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The desired product of bioprocesses is often produced in particulate form, either as an inclusion body (IB) or as a crystal. Particle harvesting is then a crucial and attractive form of product recovery. Because the liquid phase often contains other bioparticles, such as cell debris, whole cells, particulate biocatalysts or particulate by-products, the recovery of product particles is a complex process. In most cases, the particulate product is purified using selective solubilization or extraction. However, if selective particle recovery is possible, the already high purity of the particles makes this downstream process more favorable. This work gives an overview of typical bioparticle mixtures that are encountered in industrial biotechnology and the various driving forces that may be used for particle-particle separation, such as the centrifugal force, the magnetic force, the electric force, and forces related to interfaces. By coupling these driving forces to the resisting forces, the limitations of using these driving forces with respect to particle size are calculated. It shows that centrifugation is not a general solution for particle-particle separation in biotechnology because the particle sizes of product and contaminating particles are often very small, thus, causing their settling velocities to be too low for efficient separation by centrifugation. Examples of such separation problems are the recovery of IBs or virus-like particles (VLPs) from (microbial) cell debris. In these cases, separation processes that use electrical forces or fluid-fluid interfaces show to have a large potential for particle-particle separation. These methods are not yet commonly applied for large-scale particle-particle separation in biotechnology and more research is required on the separation techniques and on particle characterization to facilitate successful application of these methods in industry.

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Section: Suspension Characteristicsmentioning

confidence: 99%

Strategy for selection of methods for separation of bioparticles from particle mixtures

Hee

Hoeben

Lans

et al. 2006

Biotech & Bioengineering

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“…Bacterial Inclusion Bodies (IBs) are protein particles, ranging from 50 nm to 1 micron [1], produced by the deposition of polypeptides under cell stress conditions such as the observed in recombinant protein production processes [2]. In this situation, the strong induction of the protein expression system and the subsequent abnormal amount of newly synthesized polypeptides overload the protein quality control network driving non-folded, partially folded and properly folded proteins to aggregate through a stereospecific process [3].…”

Section: Introductionmentioning

confidence: 99%

Improving protein delivery of fibroblast growth factor-2 from bacterial inclusion bodies used as cell culture substrates

et al. 2014

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Abstract:Bacterial inclusion bodies (IBs) have been recently used to generate biocompatible cell culture interfaces with diverse effects on cultured cells such as cell adhesion enhancement, stimulation of cell growth or induction of mesenchymal stem cell differentiation. Additionally, novel applications of IBs as sustained protein delivery systems with potential applications in regenerative medicine have been successfully explored. In this scenario, with IBs gaining significance in the biomedical field, the fine tuning of this functional biomaterial is crucial. In this work, the effect of temperature on FGF-2 IB production and performance has been evaluated. FGF-2 has been overexpressed in Escherichia coli at 25 °C and 37 °C obtaining IBs with differences in size, particle structure and biological activity. Cell culture topographies made with FGF-2 IBs biofabricated at 25 °C showed higher levels of biological activity as well as a looser supramolecular structure, enabling a higher protein release from the particles. In addition, the controlled use of FGF-2 protein particles enabled the generation of functional topographies with multiple biological activities being effective on diverse cell types.

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“…Upon expression in Escherichia coli, many recombinant proteins form dense, insoluble aggregates called inclusion bodies (Taylor et al 1986). It is therefore necessary to develop efficient and scalable procedures for solubilization and refolding of recombinant proteins from inclusion bodies (Misawa and Kumagai 1999;Middelberg 2002).…”

mentioning

confidence: 99%

“…The methodology is scaleable and parallelizable and does not require subsequent concentration steps. This approach may serve as a useful complement to existing refolding strategies of diverse proteins from inclusion bodies.Keywords: three-phase partitioning; protein refolding; recombinant ribonuclease A; CcdB mutants; human CD4; inclusion bodies Supplemental material: see www.proteinscience.org Upon expression in Escherichia coli, many recombinant proteins form dense, insoluble aggregates called inclusion bodies (Taylor et al 1986). It is therefore necessary to develop efficient and scalable procedures for solubilization and refolding of recombinant proteins from inclusion bodies (Misawa and Kumagai 1999;Middelberg 2002).…”

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confidence: 99%

Refolding and simultaneous purification by three‐phase partitioning of recombinant proteins from inclusion bodies

et al. 2008

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Many recombinant eukaryotic proteins tend to form insoluble aggregates called inclusion bodies, especially when expressed in Escherichia coli. We report the first application of the technique of threephase partitioning (TPP) to obtain correctly refolded active proteins from solubilized inclusion bodies. TPP was used for refolding 12 different proteins overexpressed in E. coli. In each case, the protein refolded by TPP gave either higher refolding yield than the earlier reported method or succeeded where earlier efforts have failed. TPP-refolded proteins were characterized and compared to conventionally purified proteins in terms of their spectral characteristics and/or biological activity. The methodology is scaleable and parallelizable and does not require subsequent concentration steps. This approach may serve as a useful complement to existing refolding strategies of diverse proteins from inclusion bodies.Keywords: three-phase partitioning; protein refolding; recombinant ribonuclease A; CcdB mutants; human CD4; inclusion bodies Supplemental material: see www.proteinscience.org Upon expression in Escherichia coli, many recombinant proteins form dense, insoluble aggregates called inclusion bodies (Taylor et al. 1986). It is therefore necessary to develop efficient and scalable procedures for solubilization and refolding of recombinant proteins from inclusion bodies (Misawa and Kumagai 1999;Middelberg 2002).Refolding is typically carried out using either simple dilution into appropriate refolding conditions, dialysis or on a column (Stempfer et al. 1996;Rogl et al. 1998). Finding appropriate refolding conditions is generally the most difficult step of the purification process. Aggregation and precipitation of protein during refolding are commonly encountered problems. Factorial and other screens for refolding have been developed (Hofmann et al. 1995;Armstrong et al. 1999;Bajaj et al. 2004) to facilitate the search of appropriate refolding conditions. In the present study we describe the first application of the technique of three-phase partitioning (TPP) (Lovrien et al. 1995;Dennison and Lovrien 1997;Jain et al. 2004;Przybycien et al. 2004) to obtain active refolded proteins from solubilized inclusion bodies without any chromatographic steps. We have used TPP to refold 12 different proteins from inclusion bodies, namely, ribonuclease A Reprint requests to: Munishwar N. Gupta, Chemistry Department, Indian Institute of Technology Delhi, Hauz Khas, New Delhi 110 016, India; e-mail: munishwar48@yahoo.co.uk; fax: 91-011-26581073.Abbreviations: CcdB, controller of cell division or death B; CD4D12, first two domains of human CD4; C m , the denaturant concentration at which one half of the protein molecules are unfolded; GdmCl, guanidium chloride; IPTG, isopropyl b-D-1-thiogalactopyranoside; MBP, maltose binding protein; PTPs, protein tyrosine phosphatases; RNase A, ribonuclease A; SPR, surface plasmon resonance; TPP, three-phase partitioning; Trx, E. coli thioredoxin.Article and publication are at

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Size and Density of Protein Inclusion Bodies

Cited by 115 publications

References 9 publications

Strategy for selection of methods for separation of bioparticles from particle mixtures

Strategy for selection of methods for separation of bioparticles from particle mixtures

Improving protein delivery of fibroblast growth factor-2 from bacterial inclusion bodies used as cell culture substrates

Refolding and simultaneous purification by three‐phase partitioning of recombinant proteins from inclusion bodies

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