scholarly journals Recombinant production and purification of the human protein Tau

2018 ◽  
Vol 31 (12) ◽  
pp. 447-455 ◽  
Author(s):  
Luca Ferrari ◽  
Stefan G D Rüdiger
Keyword(s):  
Escherichia Coli ◽  
Tau Protein ◽  
High Purity ◽  
Affinity Tag ◽  
Protein Tau ◽  
Recombinant Production ◽  
N Terminus ◽  
Cytoskeletal Dynamics ◽  
Robust Protocol

Abstract Tau protein is a microtubule-stabilising protein whose aggregation is linked to Alzheimer’s Disease and other forms of dementia. Tau biology is at the heart of cytoskeletal dynamics and neurodegenerative mechanisms, making it a crucial protein to study. Tau purification, however, is challenging as Tau is disordered, which makes it difficult to produce in recombinant system and is degradation-prone. It is thus challenging to obtain pure and stable preparations of Tau. Here, we present a fast and robust protocol to purify Tau recombinantly in Escherichia coli. Our protocol allows purifying Tau either tag-less or FLAG-tagged at its N-terminus, and Tau fragments of interest. By exploiting a cleavable affinity tag and two anion exchange columns, we obtained Tau preparations of high purity, stable and suitable for in vitro studies, including aggregation experiments that resemble neurodegenerative processes.

scholarly journals Recombinant production and purification of the human protein Tau

2019 ◽  
Author(s):  
Luca Ferrari ◽  
Stefan G.D. Rüdiger
Keyword(s):  
Escherichia Coli ◽  
Tau Protein ◽  
High Purity ◽  
Affinity Tag ◽  
Protein Tau ◽  
Recombinant Production ◽  
N Terminus ◽  
Cytoskeletal Dynamics ◽  
Robust Protocol

ABSTRACTTau protein is a microtubule-stabilizing protein whose aggregation is linked to Alzheimer’s Disease and other forms of dementia. Tau biology is at the heart of cytoskeletal dynamics and neurodegenerative mechanisms, making it a crucial protein to study. Tau purification, however, is challenging as Tau is disordered, which makes it difficult to produce in recombinant system and is degradation-prone. It is thus challenging to obtain pure and stable preparations of Tau. Here, we present a fast and robust protocol to purify Tau recombinantly in Escherichia coli. Our protocol allows purifyig Tau either tag-less free or FLAG-tagged at its N-terminus. By exploiting a cleavable affinity tag and two anion exchange columns, we obtained Tau is of high purity, stable and suitable for in vitro studies, including aggregation experiments that resemble neurodegenerative processes.

scholarly journals A structure-based model for the electrostatic interaction of the N-terminus of protein tau with the fibril core of Alzheimer’s Disease filaments

2018 ◽  
Author(s):  
David R. Boyer ◽  
David S. Eisenberg
Keyword(s):  
Alzheimer’S Disease ◽  
Alzheimer's Disease ◽  
Tau Protein ◽  
Terminal Segment ◽  
Filament Formation ◽  
Binding Region ◽  
Protein Tau ◽  
Structural Details ◽  
N Terminus ◽  
Lines Of Evidence

AbstractAlthough portions of tau protein within the microtubule binding region have been shown to form the ordered core of tau filaments, the structural details of how other regions of tau participate in filament formation are so far unknown. In an attempt to understand how the N-terminus of tau may interact with fibril core, we crystallized and determined the structure of the N-terminal segment 5RQEFEV10 of tau. Several lines of evidence have shown the importance of this segment for fibril formation. The crystal structure reveals an out-of-register Class 5 steric zipper with a wet and a dry interface. To examine the possible interaction of 5RQEFEV10 with the tau fibril core, we modeled the binding of the wet interface of the 5RQEFEV10 structure with the 313VDLSKVTSKC322 region of the Alzheimer’s Disease tau filament structures. This model is consistent with, and helps to explain previous findings on the possible interaction of these two segments, distant in sequence. In addition, we discuss the possible conservation of this interaction across multiple polymorphs of tau.

scholarly journals Purification and characterization of recombinant pea-seed ferritins expressed in Escherichia coli: influence of N-terminus deletions on protein solubility and core formation in vitro

1995 ◽  
Vol 305 (1) ◽  
pp. 253-261 ◽  
Author(s):  
O Van Wuytswinkel ◽  
G Savino ◽  
J F Briat
Keyword(s):  
Escherichia Coli ◽  
Inclusion Bodies ◽  
Dna Fragments ◽  
Core Formation ◽  
Wild Type ◽  
E Coli ◽  
N Terminus ◽  
Ferroxidase Activity ◽  
Pea Seed

Plant ferritin subunits are synthesized as precursor molecules; the transit peptide (TP) in their NH2 extremity, responsible for plastid targeting, is cleaved during translocation to this compartment. In addition, the N-terminus of the mature subunit contains a plant-specific sequence named extension peptide (EP) [Ragland, Briat, Gagnon, Laulhère, Massenet, and Theil, E.C. (1990) J. Biol. Chem. 265, 18339-18344], the function of which is unknown. A novel pea-seed ferritin cDNA, with a consensus ferroxidase centre conserved within H-type animal ferritins has been characterized. This pea-seed ferritin cDNA has been engineered using oligonucleotide-directed mutagenesis to produce DNA fragments (1) corresponding to the wild-type (WT) ferritin precursor, (2) with the TP deleted, (3) with both the TP and the plant specific EP sequences deleted and (4) containing the TP but with the EP deleted. These four DNA fragments have been cloned in an Escherichia coli expression vector to produce the corresponding recombinant pea-seed ferritins. Expression at 37 degrees C led to the accumulation of recombinant pea-seed ferritins in inclusion bodies, whatever the construct introduced in E. coli. Expression at 25 degrees C in the presence of sorbitol and betaine allowed soluble proteins to accumulate when constructs with the TP deleted were used; under this condition, E. coli cells transformed with constructs containing the TP were unable to accumulate recombinant protein. Recombinant ferritins purified from inclusion bodies were found to be assembled only when the TP was deleted; however assembled ferritin under this condition had a ferroxidase activity undetectable at acid pH. On the other hand, soluble recombinant ferritins with the TP deleted and expressed at 25 degrees C were purified as 24-mers containing an average of 40-50 iron atoms per molecule. Despite the conservation in the plant ferritin subunit of a consensus ferroxidase centre, the iron uptake activity in vitro at pH 6.8 was found to be lower than that of the recombinant human H-ferritin, though it was much more active than the recombinant human L-ferritin. The recombinant ferritin with both the TP and the EP deleted (r delta TP/EP) assembled correctly as a 24-mer; it has slightly higher ferroxidase activity and decreased solubility compared with the wild-type protein with the TP deleted (r delta TP). In addition, on denaturation by urea followed by renaturation by dialysis the r delta TP/EP protein showed a 25% increase in core-formation in vitro compared with the r delta TP protein.(ABSTRACT TRUNCATED AT 400 WORDS)

scholarly journals In vitro amplification of pathogenic tau conserves disease-specific bioactive characteristics

2021 ◽  
Author(s):  
Hong Xu ◽  
Mia O’Reilly ◽  
Garrett S. Gibbons ◽  
Lakshmi Changolkar ◽  
Jennifer D. McBride ◽  
...  
Keyword(s):  
Tau Protein ◽  
Biological Activities ◽  
Corticobasal Degeneration ◽  
Tau Pathology ◽  
Protein Tau ◽  
Beta Sheet Structure ◽  
Sheet Structure ◽  
Strain Dependent ◽  
Disease Specific

AbstractThe microtubule-associated protein tau (tau) forms hyperphosphorylated aggregates in the brains of tauopathy patients that can be pathologically and biochemically defined as distinct tau strains. Recent studies show that these tau strains exhibit strain-specific biological activities, also referred to as pathogenicities, in the tau spreading models. Currently, the specific pathogenicity of human-derived tau strains cannot be fully recapitulated by synthetic tau preformed fibrils (pffs), which are generated from recombinant tau protein. Reproducing disease-relevant tau pathology in cell and animal models necessitates the use of human brain-derived tau seeds. However, the availability of human-derived tau is extremely limited. Generation of tau variants that can mimic the pathogenicity of human-derived tau seeds would significantly extend the scale of experimental design within the field of tauopathy research. Previous studies have demonstrated that in vitro seeding reactions can amplify the beta-sheet structure of tau protein from a minute quantity of human-derived tau. However, whether the strain-specific pathogenicities of the original, human-derived tau seeds are conserved in the amplified tau strains has yet to be experimentally validated. Here, we used biochemically enriched brain-derived tau seeds from Alzheimer’s disease (AD), corticobasal degeneration (CBD) and progressive supranuclear palsy (PSP) patient brains with a modified seeding protocol to template the recruitment of recombinant 2N4R (T40) tau in vitro. We quantitatively interrogated efficacy of the amplification reactions and the pathogenic fidelity of the amplified material to the original tau seeds using recently developed sporadic tau spreading models. Our data suggest that different tau strains can be faithfully amplified in vitro from tau isolated from different tauopathy brains and that the amplified tau variants retain their strain-dependent pathogenic characteristics.

scholarly journals Cortactin Recruitment by Enterohemorrhagic Escherichia coli O157:H7 during Infection In Vitro and Ex Vivo

2008 ◽  
Vol 76 (10) ◽  
pp. 4669-4676 ◽  
Author(s):  
Aurelie Mousnier ◽  
Andrew D. Whale ◽  
Stephanie Schüller ◽  
John M. Leong ◽  
Alan D. Phillips ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Actin Polymerization ◽  
Cultured Cells ◽  
Ex Vivo ◽  
Enterohemorrhagic Escherichia Coli ◽  
T3ss Effector ◽  
Mucosal Surfaces ◽  
N Terminus

ABSTRACT Enterohemorrhagic Escherichia coli (EHEC) O157:H7 is an important human pathogen that colonizes the gut mucosa via attaching and effacing (A/E) lesions; A/E lesion formation in vivo and ex vivo is dependent on the type III secretion system (T3SS) effector Tir. Infection of cultured cells by EHEC leads to induction of localized actin polymerization, which is dependent on Tir and a second T3SS effector protein, TccP, also known as EspFU. Recently, cortactin was shown to bind both the N terminus of Tir and TccP via its SH3 domain and to play a role in EHEC-triggered actin polymerization in vitro. In this study, we investigated the recruitment of cortactin to the site of EHEC adhesion during infection of in vitro-cultured cells and mucosal surfaces ex vivo (using human terminal ileal in vitro organ cultures [IVOC]). We have shown that cortactin is recruited to the site of EHEC adhesion in vitro downstream of TccP and N-WASP. Deletion of the entire N terminus of Tir or replacing the N-terminal polyproline region with alanines did not abrogate actin polymerization or cortactin recruitment. In contrast, recruitment of cortactin to the site of EHEC adhesion in IVOC is TccP independent. These results imply that cortactin is recruited to the site of EHEC adhesion in vitro and ex vivo by different mechanisms and suggest that cortactin might have a role during EHEC infection of mucosal surfaces.

scholarly journals Purification and characterisation of recombinant His-tagged RgpB gingipain from Porphymonas gingivalis

2015 ◽  
Vol 396 (4) ◽  
pp. 377-384 ◽  
Author(s):  
Florian Veillard ◽  
Barbara Potempa ◽  
Yonghua Guo ◽  
Miroslaw Ksiazek ◽  
Maryta N. Sztukowska ◽  
...  
Keyword(s):  
Affinity Chromatography ◽  
Outer Membrane ◽  
Porphyromonas Gingivalis ◽  
Virulence Factors ◽  
High Purity ◽  
Biochemical Properties ◽  
Periodontal Pathogen ◽  
Membrane Translocation ◽  
Affinity Tag

Abstract Gingipain proteases are important virulence factors from the periodontal pathogen Porphyromonas gingivalis and are the target of many in vitro studies. Due to their close biochemical properties, purification of individual gingipains is difficult and requires multiple chromatographic steps. In this study, we demonstrate that insertion of a hexahistidine affinity tag upstream of a C-terminal outer membrane translocation signal in RgpB gingipain leads to the secretion of a soluble, mature form of RgpB bearing the affinity tag that can easily be purified by nickel-chelating affinity chromatography. The final product obtained high yielding high purity is biochemically indistinguishable from the native RgpB enzyme.

Comparison of High Purity Factor IX Concentrates and a Prothrombin Complex Concentrate in a Canine Model of Thrombogenicity

1991 ◽  
Vol 66 (05) ◽  
pp. 609-613 ◽  
Author(s):  
I R MacGregor ◽  
J M Ferguson ◽  
L F McLaughlin ◽  
T Burnouf ◽  
C V Prowse
Keyword(s):  
High Purity ◽  
Time Course ◽  
Prothrombin Complex Concentrate ◽  
Degradation Products ◽  
Canine Model ◽  
Factor Ix ◽  
In Vitro Tests ◽  
Albumin Infusion

SummaryA non-stasis canine model of thrombogenicity has been used to evaluate batches of high purity factor IX concentrates from 4 manufacturers and a conventional prothrombin complex concentrate (PCC). Platelets, activated partial thromboplastin time (APTT), fibrinogen, fibrin(ogen) degradation products and fibrinopeptide A (FPA) were monitored before and after infusion of concentrate. Changes in FPA were found to be the most sensitive and reproducible indicator of thrombogenicity after infusion of batches of the PCC at doses of between 60 and 180 IU/kg, with a dose related delayed increase in FPA occurring. Total FPA generated after 100-120 IU/kg of 3 batches of PCC over the 3 h time course was 9-12 times that generated after albumin infusion. In contrast the amounts of FPA generated after 200 IU/kg of the 4 high purity factor IX products were in all cases similar to albumin infusion. It was noted that some batches of high purity concentrates had short NAPTTs indicating that current in vitro tests for potential thrombogenicity may be misleading in predicting the effects of these concentrates in vivo.

Mechanisms involved in effects of antimicrobial peptides, bactenectins ChBac3.4, ChBac5, and mini-ChBac7.5Nb, on bacterial cells

2017 ◽  
pp. 43-50
Author(s):  
О.В. Шамова ◽  
М.С. Жаркова ◽  
П.М. Копейкин ◽  
Д.С. Орлов ◽  
Е.А. Корнева
Keyword(s):  
Escherichia Coli ◽  
Antimicrobial Activity ◽  
Innate Immunity ◽  
Infectious Diseases ◽  
Metabolic Activity ◽  
Capra Hircus ◽  
Bacterial Cells ◽  
Antibiotic Resistant ◽  
Resistant Strains

Антимикробные пептиды (АМП) системы врожденного иммунитета - соединения, играющие важную роль в патогенезе инфекционных заболеваний, так как обладают свойством инактивировать широкий спектр патогенных бактерий, обеспечивая противомикробную защиту живых организмов. В настоящее время АМП рассматриваются как потенциальные соединения-корректоры инфекционной патологии, вызываемой антибиотикорезистентными бактериями (АБР). Цель данной работы состояла в изученим механизмов антибактериального действия трех пептидов, принадлежащих к семейству бактенецинов - ChBac3.4, ChBac5 и mini-ChBac7.5Nb. Эти химически синтезированные пептиды являются аналогами природных пролин-богатых АМП, обнаруженных в лейкоцитах домашней козы Capra hircus и проявляющих высокую антимикробную активность, в том числе и в отношении грамотрицательных АБР. Методы. Минимальные ингибирующие и минимальные бактерицидные концентрации пептидов (МИК и МБК) определяли методом серийных разведений в жидкой питательной среде с последующим высевом на плотную питательную среду. Эффекты пептидов на проницаемость цитоплазматической мембраны бактерий для хромогенного маркера исследовали с использованием генетически модифицированного штамма Escherichia coli ML35p. Действие бактенецинов на метаболическую активность бактерий изучали с применением маркера резазурина. Результаты. Показано, что все исследованные пептиды проявляют высокую антимикробную активность в отношении Escherichia coli ML35p и антибиотикоустойчивых штаммов Escherichia coli ESBL и Acinetobacter baumannii in vitro, но их действие на бактериальные клетки разное. Использован комплекс методик, позволяющих наблюдать в режиме реального времени динамику действия бактенецинов в различных концентрациях (включая их МИК и МБК) на барьерную функцию цитоплазматической мембраны и на интенсивность метаболизма бактериальных клеток, что дало возможность выявить различия в характере воздействия бактенецинов, отличающихся по структуре молекулы, на исследуемые микроорганизмы. Установлено, что действие каждого из трех исследованных бактенецинов в бактерицидных концентрациях отличается по эффективности нарушения целостности бактериальных мембран и в скорости подавления метаболизма клеток. Заключение. Полученная информация дополнит существующие фундаментальные представления о механизмах действия пролин-богатых пептидов врожденного иммунитета, а также послужит основой для биотехнологических исследований, направленных на разработку на базе этих соединений новых антибиотических препаратов для коррекции инфекционных заболеваний, вызываемых АБР и являющимися причинами тяжелых внутрибольничных инфекций. Antimicrobial peptides (AMPs) of the innate immunity are compounds that play an important role in pathogenesis of infectious diseases due to their ability to inactivate a broad array of pathogenic bacteria, thereby providing anti-microbial host defense. AMPs are currently considered promising compounds for treatment of infectious diseases caused by antibiotic-resistant bacteria. The aim of this study was to investigate molecular mechanisms of the antibacterial action of three peptides from the bactenecin family, ChBac3.4, ChBac5, and mini-ChBac7.5Nb. These chemically synthesized peptides are analogues of natural proline-rich AMPs previously discovered by the authors of the present study in leukocytes of the domestic goat, Capra hircus. These peptides exhibit a high antimicrobial activity, in particular, against antibiotic-resistant gram-negative bacteria. Methods. Minimum inhibitory and minimum bactericidal concentrations of the peptides (MIC and MBC) were determined using the broth microdilution assay followed by subculturing on agar plates. Effects of the AMPs on bacterial cytoplasmic membrane permeability for a chromogenic marker were explored using a genetically modified strain, Escherichia coli ML35p. The effect of bactenecins on bacterial metabolic activity was studied using a resazurin marker. Results. All the studied peptides showed a high in vitro antimicrobial activity against Escherichia coli ML35p and antibiotic-resistant strains, Escherichia coli ESBL and Acinetobacter baumannii, but differed in features of their action on bacterial cells. The used combination of techniques allowed the real-time monitoring of effects of bactenecin at different concentrations (including their MIC and MBC) on the cell membrane barrier function and metabolic activity of bacteria. The differences in effects of these three structurally different bactenecins on the studied microorganisms implied that these peptides at bactericidal concentrations differed in their capability for disintegrating bacterial cell membranes and rate of inhibiting bacterial metabolism. Conclusion. The obtained information will supplement the existing basic concepts on mechanisms involved in effects of proline-rich peptides of the innate immunity. This information will also stimulate biotechnological research aimed at development of new antibiotics for treatment of infectious diseases, such as severe in-hospital infections, caused by antibiotic-resistant strains.

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