Bovine serum albumin unfolds in Couette flow

Bekard, Innocent B.; Asimakis, Peter; Teoh, Chai Lean; Ryan, Tim; Howlett, Geoffrey J.; Bertolini, Joseph; Dunstan, Dave E.

doi:10.1039/c1sm06704d

Cited by 22 publications

(29 citation statements)

References 60 publications

(88 reference statements)

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“…Using this device, we demonstrated that extensional flow has the ability to induce the aggregation of BSA. This, together with previously published studies, suggests that while shear and extensional flow fields can both induce aggregation (11,31), their ability to do so is protein dependent. For example, both spider silk and von Willebrand factor have been observed to undergo shear-induced remodeling [which nonetheless are exposed to mixed shear/extensional flows in vivo (22,46)].…”

Section: Discussionmentioning

confidence: 83%

“…The all-α-helical, 583-residue protein BSA ( Fig. 2A) was used for our initial studies as it has well-characterized intrinsic aggregation pathways (28)(29)(30) and its behavior under shear and extensional flow fields has been investigated previously (11,31). To assess whether our extensional flow device can induce protein aggregation, 500 μL gelfiltered monodisperse BSA (Methods) at a concentration of 1, 2, 5, or 10 mg mL −1 was passed through the capillary 500, 1,000, 1,500, or 2,000 times at a plunger velocity of 8 mm s −1 (equivalent to total exposure times to extensional flow of 9, 18, 27, and 36 ms, respectively, at fixed centerline strain-(11,750 s −1 ) and (C) TEM images of 5 mg mL −1 BSA after 0 (Top) and 2,000 passes (Bottom).…”

Section: Design and Computational Characterization Of Extensional Flowmentioning

confidence: 99%

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Inducing protein aggregation by extensional flow

Dobson

Kumar

Willis

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

118

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Relative to other extrinsic factors, the effects of hydrodynamic flow fields on protein stability and conformation remain poorly understood. Flow-induced protein remodeling and/or aggregation is observed both in Nature and during the large-scale industrial manufacture of proteins. Despite its ubiquity, the relationships between the type and magnitude of hydrodynamic flow, a protein's structure and stability, and the resultant aggregation propensity are unclear. Here, we assess the effects of a defined and quantified flow field dominated by extensional flow on the aggregation of BSA, β 2 -microglobulin (β 2 m), granulocyte colony stimulating factor (G-CSF), and three monoclonal antibodies (mAbs). We show that the device induces protein aggregation after exposure to an extensional flow field for 0.36-1.8 ms, at concentrations as low as 0.5 mg mL −1 . In addition, we reveal that the extent of aggregation depends on the applied strain rate and the concentration, structural scaffold, and sequence of the protein. Finally we demonstrate the in situ labeling of a buried cysteine residue in BSA during extensional stress. Together, these data indicate that an extensional flow readily unfolds thermodynamically and kinetically stable proteins, exposing previously sequestered sequences whose aggregation propensity determines the probability and extent of aggregation.extensional flow | aggregation | unfolding | bioprocessing | antibody P roteins are dynamic and metastable and consequently have conformations that are highly sensitive to the environment (1). Over the last 50 y the effect of changes in temperature, pH, and the concentration of kosmatropic/chaotropic agents on the conformational energy landscape of proteins has become well understood (1). This, in turn, has allowed a link to be established between the partial or full unfolding of proteins and their propensity to aggregate (2). The force applied onto a protein as a consequence of hydrodynamic flow has also been observed to trigger protein aggregation and has fundamental (3), medical (4), and industrial relevance, especially in the manufacture of biopharmaceuticals (5-8). Although a wealth of studies have been performed (7,(9)(10)(11)(12)(13), no consensus has emerged on the ability of hydrodynamic flow to induce protein aggregation (7,14,15). This is due to the wide variety of proteins used (ranging from lysozyme, BSA, and alcohol dehydrogenase to IgGs), differences in the type of flow field generated (e.g., shear, extensional, or mixtures of these), and to the presence or absence of an interface (16). A shearing flow field (Fig. 1A, Top) is caused by a gradient in velocity perpendicular to the direction of travel and is characterized by the shear rate (s −1 ). This results in a weak rotating motion of a protein alongside translation in the direction of the flow. An extensional flow field (Fig. 1A, Bottom) is generated by a gradient in velocity in the direction of travel and is characterized by the strain rate (s −1 ). A protein in this type of flow would experien...

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Section: Discussionmentioning

confidence: 83%

Section: Design and Computational Characterization Of Extensional Flowmentioning

confidence: 99%

Inducing protein aggregation by extensional flow

Dobson

Kumar

Willis

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

118

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Section: Introductionmentioning

confidence: 98%

“…Many proteins are submitted to mechanical shearing through different food processes such as filtration, mixing, pumping and extrusion (Ker & Chen, 1998). Shearing has been shown to influence the structural conformation of proteins and change the functional and gelation properties of proteins (Ker & Chen, 1998;Dunstan et al, 2009a;Bekard et al, 2012;Ow & Dunstan, 2013). Mechanical shearing may modify chemical and enzymatic treatments during processing and may also improve functional properties of proteins in specific conditions (Ker & Chen, 1998).…”

Section: Introductionmentioning

confidence: 99%

“…The effects of shear on conformation and properties of different globular protein have been extensively investigated. At low pH, the protein is highly charged and the application of shear stress promotes the formation of fibrillar structure in globular proteins such as whey protein isolate (Akkermans et al, 2008), Bovine serum albumin (Bekard et al, 2012), lysozyme (Ow & Dunstan, 2013), b-lactoglobulin (Akkermans et al, 2006;Hill et al, 2006;Dunstan et al, 2009b), amyloid-b (Hamilton-Brown et al, 2008Dunstan et al, 2009a) and soy proteins (Ker & Chen, 1998;Akkermans et al, 2007). showed that heavily sheared whey protein concentrate formed weak gels with a lower storage modulus than that of the unsheared gels, while the microstructure of shear treated and unsheared gels did not show significant structural differences as observed optically.…”

Section: Introductionmentioning

confidence: 99%

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The effect of shear on the rheology and structure of heat‐induced whey protein gels

Badii

Atri

Dunstan

2016

Int J of Food Sci Tech

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The influence of mechanical shearing on the small deformation properties and microstructure of heat-induced whey protein gel has been studied. The viscoelastic properties of these gels at different concentrations of 10% and 20% (w/w) exposed to different shear rates of 0, 50, 100, 200 and 500 s À1 during gelation were measured using dynamic oscillatory rheometry. The structure of both the shear treated and unsheared gels was then investigated using light microscopy. The results showed that the storage modulus of the gels at both concentrations was increased by increasing the shear rate exposure during gelation while the shear-treated gels were more elastic and showed frequency-independent behaviour. As the total protein concentration of the gel increased, the viscoelastic properties of the gels also increased significantly and the gels showed greater elasticity. The gels obtained from the higher shear rate exposure were stronger with higher elastic moduli at both protein concentrations. Images of the gels obtained using light microscopy showed that shearing resulted in phase separation and some aggregation in the structure of the gels at both concentrations. However, the shearing rates applied in this study were not enough to cause aggregation breakdown in the gel network.

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Effect of characteristics of shear force on secondary structures and viscosity of bovine serum albumin solution

Sharma

Pattanayek

2018

Rheol Acta

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Bovine serum albumin unfolds in Couette flow

Cited by 22 publications

References 60 publications

Inducing protein aggregation by extensional flow

Inducing protein aggregation by extensional flow

The effect of shear on the rheology and structure of heat‐induced whey protein gels

Effect of characteristics of shear force on secondary structures and viscosity of bovine serum albumin solution

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