Differential Precipitation and Solubilization of Proteins

Ryan, Barry J.

doi:10.1007/978-1-4939-6412-3_10

Cited by 9 publications

(10 citation statements)

References 38 publications

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“…The cause of protein precipitation remains unknown, but possible explanations include natural degradation over 80 years, oxidation, and change in salt concentrations and/or pH. 16 Plasma is superior to serum for haemorrhage management as it contains serum proteins, such as albumin for volume expansion to maintain organ perfusion, as well as fibrinogen and other important clotting factors crucial for attenuating bleeding. The lack of clotting proteins in serum can be advantageous for prolonged storage, including freeze/thaw cycles.…”

Section: Discussionmentioning

confidence: 99%

“…Despite significant protein precipitates forming during the reconstitution of the 1943 sample, we were able to remove the precipitates through centrifugation and filtration. The cause of protein precipitation remains unknown, but possible explanations include natural degradation over 80 years, oxidation, and change in salt concentrations and/or pH 16 …”

Section: Discussionmentioning

confidence: 99%

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Past meets present: Reviving 80‐year‐old Canadian dried serum from World War II and its significance in advancing modern freeze‐dried plasma for prehospital management of haemorrhage

Singh,

Peng,

Moes

et al. 2024

Br J Haematol

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SummaryDuring World War II, Charles H. Best utilized Charles R. Drew's plasma isolation and drying technique to lead Canada's initiative to provide dried serum as a means of primary resuscitation for British casualties on the frontlines. Serum was likely utilized over plasma for its volume expansion properties without the risk of clotting during prolonged storage. We reconstituted dried serum from 1943 and discovered intact albumin, as well as anti‐thrombin, plasminogen, protein C and protein S activity. Proteomic analysis identified 71 proteins, most prominent being albumin, and positive for hepatitis B by serological testing. Transmission of blood‐borne diseases ended the programme, until modern advances in testing and pathogen reduction revived this technology. We tested the latest iteration of Canadian freeze‐dried plasma (FDP), which was stored for 4 years, and demonstrated that its clotting capacity remained equivalent to fresh frozen plasma. We recommend that FDP is a strong alternative to contemporary prehospital resuscitation fluids (e.g. normal saline/lactated Ringer's) in managing prehospital haemorrhage where whole blood is unavailable.

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Past meets present: Reviving 80‐year‐old Canadian dried serum from World War II and its significance in advancing modern freeze‐dried plasma for prehospital management of haemorrhage

Singh,

Peng,

Moes

et al. 2024

Br J Haematol

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“…In incubated plates, significant growth inhibition was observed for all purified protein concentrations (25,35,45, and 50 µg). No visible mycelial growth was seen after 24 h due to its slow growth rate [24]. On culture plates, the mycelial mass ring continued to increase proportionally with time in control well; however, a clear inhibitory effect of recombinant protein with concentrations of 25, 35, 45, and 50 µg was observed after 48, 72, 96, 120 and 144 h of C. canescens culturing (Fig.…”

Section: Antifungal Activity Of Rmb-clsrgmentioning

confidence: 92%

“…The pETMBRg clone (MB-CLsRG/ pET28a) was cultured on LB broth supplemented with isopropyl-β-D-thiogalactopyranoside (IPTG) for inducing recombinant protein expression, and 100 μg/mL kanamycin by following the protocol described by Boshtam et al [22]. The purification of expressed recombinant protein (MBRg protein) was performed by adopting the protocols described by Zandvakili et al [23] and Ryan [24]. The purified expressed protein was run on Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and visualized by Coomassie Brilliant Blue R 250 (ROTH) protein stain.…”

Section: Expression and Purification Of Recombinant Proteinmentioning

confidence: 99%

Development of Molecular Marker Linked with Cercospora Leaf Spot (CLS) Disease Resistance in Vigna radiata, Cloning, and Expression for Evaluating Antifungal Activity against Cercospora canescens

Babar¹,

Ijaz²,

Haq³

et al. 2023

Phyton

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We developed a molecular marker for MAS of mungbean resistant varieties against CLS from the consensus sequence (MB-CLsRG) of identified RGAs (MB-ClsRCaG1 and MB-ClsRCaG2). The MB-CLsRG sequence-specific primer pair was used to screen Cercospora leaf spot (CLS) resistant varieties of mungbean in genomic analysis that showed congruency with phenotypic screening. Validation of molecular marker linkage with CLS resistance was performed using rtPCR in transcriptomic analysis. The sequenced PCR products showed 100% homology with MB-CLsRG sequence and putative disease resistance proteins that confirmed the linkage of molecular marker with CLS resistance in mungbean. The antifungal potential of MB-CLsRG gene encoding protein was assessed. The MB-CLsRG gene sequence was cloned in the E. coli expression vector for recombinant protein production. The recombinant protein was then investigated for its in vitro antifungal potential against Cercospora canescens. The in vitro investigation showed strong antifungal activity of recombinant protein as it restricted the growth of fungal mycelial mass. The results validated the linkage of developed marker with CLS-resistant mungbean varieties; therefore, it can be used to screen resistant varieties from a large population in MAS. Moreover, the recombinant protein of the MB-CLsRG gene sequence revealed antifungal potential, which proved the gene sequence could be suitable to use in transgenic plants technology to develop fungal-resistant transgenic crops.

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“…Before processing the target proteins into various formats, these harsh denaturants must be removed completely. Generally, solubilization of IBs with high concentration of denaturing reagents often leads to poor recovery of bioactive species, and large amount of precipitations form during the refolding process [31,32].…”

Section: Introductionmentioning

confidence: 99%

One-step heating strategy for efficient solubilization of recombinant spider silk protein from inclusion bodies

Cai

Chen

et al. 2020

BMC Biotechnol

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Background: Spider silk is a proteinaceous fiber with remarkable mechanical properties spun from spider silk proteins (spidroins). Engineering spidroins have been successfully produced in a variety of heterologous hosts and the most widely used expression system is Escherichia coli (E. coli). So far, recombinantly expressed spidroins often form insoluble inclusion bodies (IBs), which will often be dissolved under extremely harsh conditions in a traditional manner, e.g. either 8 mol/L urea or 6 mol/L guanidine hydrochloride, highly risking to poor recovery of bioactive proteins as well as unexpected precipitations during dialysis process. Results: Here, we present a mild solubilization strategy-one-step heating method to solubilize spidroins from IBs, with combining spidroins' high thermal stability with low concentration of urea. A 430-aa recombinant protein (designated as NM) derived from the minor ampullate spidroin of Araneus ventricosus was expressed in E. coli, and the recombinant proteins were mainly present in insoluble fraction as IBs. The isolated IBs were solubilized parallelly by both traditional urea-denatured method and one-step heating method, respectively. The solubilization efficiency of NM IBs in Tris-HCl pH 8.0 containing 4 mol/L urea by one-step heating method was already comparable to that of 7 mol/L urea with using traditional urea-denatured method. The effects of buffer, pH and temperature conditions on NM IBs solubilization of one-step heating method were evaluated, respectively, based on which the recommended conditions are: heating temperature 70-90°C for 20 min, pH 7.0-10, urea concentration 2-4 mol/L in normal biological buffers. The recombinant NM generated via the one-step heating method held the potential functions with self-assembling into sphere nanoparticles with smooth morphology. Conclusions: The one-step heating method introduced here efficiently solubilizes IBs under relatively mild conditions compared to the traditional ones, which might be important for the downstream applications; however, this protocol should be pursued carefully in terms of urea-induced modification sensitive applications. Further, this method can be applied under broad buffer, pH and temperature conditions, conferring the potential to apply to other thermal stable proteins.

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Differential Precipitation and Solubilization of Proteins

Cited by 9 publications

References 38 publications

Past meets present: Reviving 80‐year‐old Canadian dried serum from World War II and its significance in advancing modern freeze‐dried plasma for prehospital management of haemorrhage

Past meets present: Reviving 80‐year‐old Canadian dried serum from World War II and its significance in advancing modern freeze‐dried plasma for prehospital management of haemorrhage

Development of Molecular Marker Linked with Cercospora Leaf Spot (CLS) Disease Resistance in Vigna radiata, Cloning, and Expression for Evaluating Antifungal Activity against Cercospora canescens

One-step heating strategy for efficient solubilization of recombinant spider silk protein from inclusion bodies

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