Long‐Term Storage of Proteins

Carpenter, John F.; Manning, Mark C.; Randolph, Theodore W.

doi:10.1002/0471140864.ps0406s27

Cited by 29 publications

(20 citation statements)

References 21 publications

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“…Indeed, some molecules appeared to have adopted a more compact structure (migrating slightly faster than the native α 2 M band), while others had formed smaller species (based on the relative mobility of these species they are likely to be dimers, lower band in lane 3) and still others had formed higher molecular weight species (due to non-covalent self-association of the protein). As a commonly used strategy for long-term storage of proteins involves freezing [ 51 ], the protein was analyzed after storage in PBS/Az at -20°C for 10 days; less than 50% of the purified α 2 M was found to migrate to a position corresponding to native α 2 M when assessed by native PAGE, the remainder of the protein migrated to a position corresponding to transformed α 2 M ( Fig 1B ). The extent of α 2 M transformation was found to be reduced, but not abolished, by rapid freezing of the protein in liquid nitrogen (-196°C) prior to storage at -20°C ( Fig 1B ).…”

Section: Resultsmentioning

confidence: 99%

Alpha-2-Macroglobulin Is Acutely Sensitive to Freezing and Lyophilization: Implications for Structural and Functional Studies

et al. 2015

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Alpha-2-macroglobulin is an abundant secreted protein that is of particular interest because of its diverse ligand binding profile and multifunctional nature, which includes roles as a protease inhibitor and as a molecular chaperone. The activities of alpha-2-macroglobulin are typically dependent on whether its conformation is native or transformed (i.e. adopts a more compact conformation after interactions with proteases or small nucleophiles), and are also influenced by dissociation of the native alpha-2-macroglobulin tetramer into stable dimers. Alpha-2-macroglobulin is predominately present as the native tetramer in vivo; once purified from human blood plasma, however, alpha-2-macroglobulin can undergo a number of conformational changes during storage, including transformation, aggregation or dissociation. We demonstrate that, particularly in the presence of sodium chloride or amine containing compounds, freezing and/or lyophilization of alpha-2-macroglobulin induces conformational changes with functional consequences. These conformational changes in alpha-2-macroglobulin are not always detected by standard native polyacrylamide gel electrophoresis, but can be measured using bisANS fluorescence assays. Increased surface hydrophobicity of alpha-2-macroglobulin, as assessed by bisANS fluorescence measurements, is accompanied by (i) reduced trypsin binding activity, (ii) increased chaperone activity, and (iii) increased binding to the surfaces of SH-SY5Y neurons, in part, via lipoprotein receptors. We show that sucrose (but not glycine) effectively protects native alpha-2-macroglobulin from denaturation during freezing and/or lyophilization, thereby providing a reproducible method for the handling and long-term storage of this protein.

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Section: Resultsmentioning

confidence: 99%

Alpha-2-Macroglobulin Is Acutely Sensitive to Freezing and Lyophilization: Implications for Structural and Functional Studies

et al. 2015

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“…FFP is a complex biological material which contains a plethora of bioactive molecules (18). Although optimal conditions for storage are distinctive for each protein, it is well known that proteins generally alter structural integrity and activity as a result of refrigeration due to proteolysis, aggregation, and suboptimal buffer conditions (19). We have recently shown that refrigeration of FFP decreased its coagulation factors and diminished its beneficial effects on endothelial function and resuscitation in an animal model of HS (10).…”

Section: Introductionmentioning

confidence: 99%

Increased Transforming Growth Factor β Contributes to Deterioration of Refrigerated Fresh Frozen Plasma's Effects In Vitro on Endothelial Cells

Duan

Cao²,

Deng

et al. 2011

Shock

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Resuscitation with fresh frozen plasma (FFP) is associated with improved outcomes after hemorrhagic shock (HS). Many trauma centers are using thawed plasma which has been stored for up to 5 days at 4°C (refrigeration), yet the effect of refrigeration on FFP is relatively unknown. Previously our group showed that refrigeration of FFP changed its coagulation factors and diminished its beneficial effects on endothelial cell (EC) function and resuscitation in an animal model of HS.We hypothesize that growth factor composition of FFP is altered during refrigeration, leading to a diminished beneficial effect on EC. Transforming growth factor (TGF)-β is a potent inhibitor of EC migration and is released during refrigeration of platelets. We found increased TGF-β1 protein levels and greater activation of downstream mediators Smad2/3 during refrigeration of FFP. Both Day 0 FFP (used on the same day after being thawed) and Day 5 FFP (used after being thawed and refrigerated for 5 days) stimulated EC migration in vitro; however, the EC migration in Day 5 FFP was significantly reduced. Inhibition of TGF-β type I receptor blocked FFP-induced Smad3 signaling in EC cells and restored the effectiveness of Day 5 FFP on EC migration to a comparable level seen in Day 0 FFP. These data suggest that the increased TGF-β levels during FFP refrigeration contribute to the deterioration of refrigerated FFP's effects on EC migration. This study identifies a novel molecular mechanism contributing to the reduced efficacy of refrigerated FFP.

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“…Although optimal conditions for storage are distinctive to each protein, it is well known that proteins generally lose structural integrity and activity as a result of storage at 4°C because of proteolysis, aggregation, and suboptimal buffer conditions. 24 Consequently, to prevent rapid degradation of proteins, preserve function, and prevent variable results, standard techniques in the basic science laboratories mandates –80°C storage of proteins used in experiments.…”

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confidence: 99%

Protective Effects of Fresh Frozen Plasma on Vascular Endothelial Permeability, Coagulation, and Resuscitation After Hemorrhagic Shock Are Time Dependent and Diminish Between Days 0 and 5 After Thaw

Pati

Matijevic

Doursout

et al. 2010

Journal of Trauma: Injury, Infection &Amp; Critical Care

157

146

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Background Clinical studies have shown that resuscitation with fresh frozen plasma (FFP) is associated with improved outcome after severe hemorrhagic shock (HS). We hypothesized that in addition to its effects on hemostasis, FFP has protective and stabilizing effects on the endothelium that translate into diminished endothelial cell (EC) permeability and improved resuscitation in vivo after HS. We further hypothesized that the beneficial effects of FFP would diminish over 5 days of routine storage at 4°C. Methods EC permeability was induced by hypoxia and assessed by the passage of 70-kDa Dextran between monolayers. Thrombin generation time and coagulation factor levels or activity were assessed in FFP. An in vivo rat model of HS and resuscitation was used to determine the effects of FFP on hemodynamic stability. Results Thawed FFP inhibits EC permeability in vitro by 10.2-fold. Protective effects diminish (to 2.5-fold) by day 5. Thrombin generation time is increased in plasma that has been stored between days 0 and 5. In vivo data show that day 0 FFP is superior to day 5 FFP in maintaining mean arterial pressure in rats undergoing HS with resuscitation. Conclusion Both in vitro and in vivo studies show that FFP has beneficial effects on endothelial permeability, vascular stability, and resuscitation in rats after HS. The benefits are independent of hemostasis and diminish between days 0 and 5 of storage.

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Long‐Term Storage of Proteins

Cited by 29 publications

References 21 publications

Alpha-2-Macroglobulin Is Acutely Sensitive to Freezing and Lyophilization: Implications for Structural and Functional Studies

Alpha-2-Macroglobulin Is Acutely Sensitive to Freezing and Lyophilization: Implications for Structural and Functional Studies

Increased Transforming Growth Factor β Contributes to Deterioration of Refrigerated Fresh Frozen Plasma's Effects In Vitro on Endothelial Cells

Protective Effects of Fresh Frozen Plasma on Vascular Endothelial Permeability, Coagulation, and Resuscitation After Hemorrhagic Shock Are Time Dependent and Diminish Between Days 0 and 5 After Thaw

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