The authors would like to acknowledge Natalie Doude and David Westerway (University of Alberta) for providing us Sho cDNA, Christophe Chevalier (INRA) for plasmid construction, Michel Br&#233;mont (INRA) for providing us BL21Sol bacteria; Christine Longin (INRA) for TEM assistance and Edith Pajot-Augy (INRA) for comments and proofreading.

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<li>Sorensen, H. P., Mortensen, K. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Advanced+genetic+strategies+for+recombinant+protein+expression+in+Escherichia+coli%5BTitle%5D)+AND+%22Journal+of+Biotechnology%22%5BJournal%5D)">Advanced genetic strategies for recombinant protein expression in Escherichia coli.</a> Journal of Biotechnology. 115, 113-128 (2005).</li>
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<li>Englund, P. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+structure+and+biosynthesis+of+glycosyl+phosphatidylinositol+protein+anchors%5BTitle%5D)+AND+%22Annual+Review+of+Biochemistry%22%5BJournal%5D)">The structure and biosynthesis of glycosyl phosphatidylinositol protein anchors.</a> Annual Review of Biochemistry. 62, 121-138 (1993).</li>
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The authors have nothing to disclose.

ويرد بروتوكول سهلة وفعالة لكلا التعبير وتنقية كمية كبيرة من الماوس المؤتلف البروتين Shadoo. الطريقة الموصوفة يسمح ذوبان ناجحة وتنقية (صاحب 6) -tagged البروتين Shadoo المؤتلف. من المهم، كما ورد في العنوان، وأنه عندما أعرب في بكتيريا E. بدائيات النواة القولونية Sha...

أمراض الاعصاب بريون، التي تشمل البقري مرض جنون البقر في، سكرابي في الأغنام ومرض كروتزفيلد جاكوب في البشر، هي قاتلة وغير قابل للشفاء. تتميز أمراض البريون من قبل التغييرات متعلق بتكوين من بروتين بريون الخلوي تتكون أساسا من α-اللوالب، إلى منطقة β ورقة عبر التخصيب اميلويد الملتزم 1،2. عبر β-هياكل تحتوي على المكتظ وأمر غاية β اوراق مثل البلورية استقرت مع الروابط الهيدروجينية 3،4. amyloids بريون يمكن نسخ الذاتي المناسب إلى خيوط β على حافة المتزايدة التي توفر نموذجا لتوظيف وتحويل وحدة البروتين أحادى. وفقا لفرضية &quot;البروتين فقط&quot;، والملتزم اميلويد من بروتين بريون هي الوكيل الوحيد المعدية. ومع ذلك، فقد تم اقتراح بعض الجزيئات الحيوية الأخرى على أنها ضرورية لاضطرابات بريون. على سبيل المثال، يعتقد أن أغشية الخلايا لتكون مكانا حيث conversiعلى سيقام وقد أظهرت بعض الدهون سالبة الشحنة لتعزيز 5،6 عملية التحويل. وبالإضافة إلى ذلك، قد تكون بعض البروتينات أيضا تشارك في أمراض البريون. على وجه التحديد، هناك اثنين آخرين من أعضاء الأسر من البروتين بريون: Doppel وShadoo 7،8. Doppel لديها بنية جامدة نسبيا استقر مع الجسور دى كبريتيد وفشل لبلمرة الذات ومجموع المباراتين لييفات اميلويد 9. في المقابل، على غرار بروتين بريون، يمكن Shadoo اعتماد هيكل المخصب ورقة β-والزميلة الذاتي في الهياكل ييف اميلويد. تبين أن Shadoo يمكن fibrillate في ظل ظروف الأم أو على الأغشية ملزمة سالبة الشحنة 10،11. تحويل Shadoo إلى ألياف تشبه اميلويد قد تترافق مع أمراض. في الواقع، وغير مفهومة الآليات التي هياكل تشبه اميلويد تسبب المرض. Shadoo يتحمل مجال مسعور (HD)، وسلسلة من جنبا إلى جنب الارجنتين / الغليسين يكرر ر مماثلس الجزء N-محطة للبروتين بريون (الشكل 1A). كما-N محطة جزء من بروتين بريون، وغاية اتهم Shadoo بشكل إيجابي ويبدو أن غير منظم أصلا البروتين 11،12. يبدو Shadoo أن تكون ذات صلة وظيفيا إلى اضطرابات بريون أنه يمكن ربط مباشرة بروتين بريون. بالإضافة إلى ذلك، ينظم التعبير إلى أسفل أثناء بريون أمراض 13،14. ومع ذلك، لم يثبت حتى الآن دور Shadoo في مرض بريون. وضعنا البلازميد التي تحمل تسلسل ترميز الجين الماوس Shadoo. تم استخدام البلازميد لتحويل E. القولونية لإنتاج N-صاحب محطة 6 الانصهار البروتين Shadoo. تم تأسيس هذا النظام التعبير بشكل جيد في مختبرنا ويستخدم عادة في مشاريعنا الجارية 6،15،16. قضية مهمة للتعبير عن Shadoo هو اختيار E. سلالة القولونية. بينما يستخدم BL21 المختصة سلالة بكتيرية عادة للتعبير عن المؤيد المؤتلفوأعرب عن البروتينات Shadoo بنجاح وتنقيته فقط من سلالة بكتيرية تحول SoluBL21. SoluBL21 E. المختصة القولونية هي متحولة تحسين BL21 سلالة المضيف المتقدمة لإنتاج البروتينات التي التعبير في BL21 الأم أعطى أي منتج قابل للذوبان يمكن كشفها. التعبير عن مستوى عال من Shadoo في SoluBL21 E. القولونية يؤدي إلى تراكم البروتين في الهيئات إدراج. كسمة عامة، عندما يتم التعبير عن درجة عالية من بروتين غير الناطقين بها في E. القولونية، هذا البروتين يميل إلى تتراكم في الهيئات إدراج غير قابلة للذوبان. Shadoo ربما المجاميع من خلال التفاعلات غير التساهمية مسعور أو الأيونية (أو مزيج من الاثنين معا) إلى البنى الدينامية التخصيب التي شكلتها البروتين مطوية بدرجات متفاوتة. ونتيجة لذلك، تتكون تنقية اثنين على الأقل من الخطوات التالية: (أ) فصل انتقائي من البروتين من الجزيئات الحيوية الأخرى في وسط تمسخ، و (ثانيا) من استعادة الطبيعة من البروتين النقي يستخدم في المختبر للطيالتقنيات. وقد تحقق الفصل الانتقائي للShadoo في حل العازلة التي تحتوي على اليوريا 8M (أو بدلا من ذلك 6M الجوانيدين حمض الهيدروكلوريك). ويمكن أن يتم إزالة اليوريا واستعادة الطبيعة من البروتين عن طريق تطبيق بروتوكولات مختلفة: (ط) استعادة الطبيعة في محلول درجة الحموضة الحمضية للحصول على غير منظم البروتين Shadoo أحادى، أو (ب) استعادة الطبيعة في حل من الرقم الهيدروجيني ≥ 7 للحصول على Shadoo بلمرة إلى ألياف الأميلويد مستقرة بزخارف عبر β ورقة مميزة.

<table><tbody><tr><td>AKTA FPLC&amp;nbsp;</td><td>GE, Amersham</td><td># 1363</td><td></td></tr><tr><td>HiLoad 16/60 Superdex</td><td>GE Healthcare</td><td>17-1069-01</td><td></td></tr><tr><td>HiTrap IMAC</td><td>GE Healthcare</td><td>GE17-0920-05</td><td></td></tr><tr><td>HiPrep26/10 Desalting column</td><td>GE Healthcare</td><td>GE17-5087-01</td><td></td></tr><tr><td>SoluBL21 Competent E. coli</td><td>Genlantis</td><td>C700200</td><td></td></tr><tr><td>pET-28b+</td><td>Novogen</td><td>69865-3</td><td></td></tr><tr><td>IPTG</td><td>Sigma-Aldrich</td><td>I6758</td><td></td></tr><tr><td>LB-Broth medium</td><td>Sigma-Aldrich</td><td>L3022</td><td></td></tr><tr><td>Eppendorf Biophotometar</td><td>Eppendorf</td><td>550507804</td><td></td></tr><tr><td>Trito X-100</td><td>Life Technologies</td><td>85111</td><td></td></tr><tr><td>NiSO4</td><td>Sigma-Aldrich</td><td>656895</td><td></td></tr><tr><td>EDTA</td><td>Sigma-Aldrich</td><td>E6758</td><td></td></tr><tr><td>Tris-HCl</td><td>Sigma-Aldrich</td><td>RES3098T</td><td></td></tr><tr><td>Protease Inhibitor Cocktail Tablets</td><td>Roche</td><td>4693116001</td><td></td></tr><tr><td>NaCl</td><td>Sigma-Aldrich</td><td>746398</td><td></td></tr><tr><td>Imidazol</td><td>Sigma-Aldrich</td><td>I5513</td><td></td></tr><tr><td>Gdn-HCl</td><td>Sigma-Aldrich</td><td>G4505</td><td></td></tr><tr><td>protein size marker&amp;nbsp;</td><td>Thermo Fisher Scientific</td><td>26620</td><td></td></tr></tbody></table>

purification and refolding to amyloid fibrils of (his)6-tagged recombinant shadoo protein expressed as inclusion bodies in e. coli

نظام كولاي القولونية التعبير هو أداة قوية لإنتاج بروتينات حقيقية النواة المؤتلف. نستخدمها لإنتاج Shadoo، وهو بروتين عائلة بريون. وأعرب طريقة الكروماتوغرافي لتنقية (صاحب) 6 -tagged Shadoo المؤتلف كما هو وصفها الهيئات إدراج. وsolubilized الهيئات إدراجها في 8 M اليوريا وملزمة لني 2+ العمود -charged لأداء أيون التقارب اللوني. ومزال البروتينات ملزمة متدرجة من إميدازول. يتعرض الكسور التي تحتوي على البروتين Shadoo إلى استبعاد حجم اللوني للحصول على بروتين عالي النقاء. في خطوة تنقية النهائي Shadoo والمحلاة لإزالة الأملاح واليوريا وإميدازول. البروتين Shadoo المؤتلف هو كاشف المهم للدراسات الفيزيائية الحيوية والكيميائية الحيوية من اضطرابات التشكل البروتين التي تحدث في أمراض البريون. أظهرت العديد من التقارير أن الأمراض العصبية بريون تنشأ من ترسب طعنةلو أمرت الألياف اميلويد. / ترد بالوسط الأساسية عينة البروتوكولات التي تصف كيفية fibrillate Shadoo إلى الألياف اميلويد في الحمضية ومحايدة. الطرق حول كيفية إنتاج وfibrillate Shadoo يمكن أن تسهل البحوث في المختبرات العاملة في مجال الأمراض بريون، لأنه يسمح لإنتاج كميات كبيرة من البروتين بطريقة سريعة ومنخفضة التكلفة.

يتم الحصول على حوالي 5-10 ملغ من البروتين المنقى Shadoo للتر الواحد من ثقافة البكتيرية. وهناك حاجة إلى تنقية من خطوتين للحصول على Shadoo المؤتلف عالية النقاء. يتم تنفيذ الخطوة الأولى من قبل اللوني تقارب مع العمود -charged ني 2+ أن يحتفظ صاحب-البروتينات الموسومة. يتعرض كسور م...

Watch this Scientific Journal Video about Purification and Refolding to Amyloid Fibrils of His6-tagged Recombinant Shadoo Protein Expressed as Inclusion Bodies in E. coli at JoVE.com

Purification and Refolding to Amyloid Fibrils of His6-tagged Recombinant Shadoo Protein Expressed as Inclusion Bodies in E. coli

A two-step chromatographic method is described for the purification of recombinant Shadoo protein expressed as inclusion bodies in Escherichia coli, as well as a protocol to fibrillate purified Shadoo into amyloid structures.

تنقية وRefolding إلى اميلويد الألياف من (صاحب)<sub&gt; 6</sub&gt; -tagged المؤتلف Shadoo البروتين التعبير عن الهيئات إدراج في<em&gt; E. القولونية</em

The Escherichia coli expression system is a powerful tool for the production of recombinant eukaryotic proteins. We use it to produce Shadoo, a protein ...

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Purification and Refolding to Amyloid Fibrils of (His)6-tagged Recombinant Shadoo Protein Expressed as Inclusion Bodies in E. coli

14.1K Views.  INRA. A two-step chromatographic method is described for the purification of recombinant Shadoo protein expressed as inclusion bodies in Escherichia coli, as well as a protocol to fibrillate purified Shadoo into amyloid structures. 

Video: Purification and Refolding to Amyloid Fibrils of His6-tagged Recombinant Shadoo Protein Expressed as Inclusion Bodies in E. coli

Selection of Aptamers for Amyloid &beta;-Protein, the Causative Agent of Alzheimer&#39;s Disease

Alzheimer's disease (AD) is a progressive, age-dependent, neurodegenerative disorder with an insidious course that renders its presymptomatic diagnosis difficult1. Definite AD diagnosis is achieved only postmortem, thus establishing presymptomatic, early diagnosis of AD is crucial for developing and administering effective therapies2,3.
Amyloid &beta;-protein (A&beta;) is central to AD pathogenesis. Soluble, oligomeric A&beta; assemblies are believed to affect neurotoxicity underlying synaptic dysfunction and neuron loss in AD4,5. Various forms of soluble A&beta; assemblies have been described, however, their interrelationships and relevance to AD etiology and pathogenesis are complex and not well understood6. Specific molecular recognition tools may unravel the relationships amongst A&beta; assemblies and facilitate detection and characterization of these assemblies early in the disease course before symptoms emerge. Molecular recognition commonly relies on antibodies. However, an alternative class of molecular recognition tools, aptamers, offers important advantages relative to antibodies7,8. Aptamers are oligonucleotides generated by in-vitro selection: systematic evolution of ligands by exponential enrichment (SELEX)9,10. SELEX is an iterative process that, similar to Darwinian evolution, allows selection, amplification, enrichment, and perpetuation of a property, e.g., avid, specific, ligand binding (aptamers) or catalytic activity (ribozymes and DNAzymes).
Despite emergence of aptamers as tools in modern biotechnology and medicine11, they have been underutilized in the amyloid field. Few RNA or ssDNA aptamers have been selected against various forms of prion proteins (PrP)12-16. An RNA aptamer generated against recombinant bovine PrP was shown to recognize bovine PrP-&beta;17, a soluble, oligomeric, &beta;-sheet-rich conformational variant of full-length PrP that forms amyloid fibrils18. Aptamers generated using monomeric and several forms of fibrillar &beta;2-microglobulin (&beta;2m) were found to bind fibrils of certain other amyloidogenic proteins besides &beta;2m fibrils19. Ylera et al. described RNA aptamers selected against immobilized monomeric A&beta;4020. Unexpectedly, these aptamers bound fibrillar A&beta;40. Altogether, these data raise several important questions. Why did aptamers selected against monomeric proteins recognize their polymeric forms? Could aptamers against monomeric and/or oligomeric forms of amyloidogenic proteins be obtained? To address these questions, we attempted to select aptamers for covalently-stabilized oligomeric A&beta;4021 generated using photo-induced cross-linking of unmodified proteins (PICUP)22,23. Similar to previous findings17,19,20, these aptamers reacted with fibrils of A&beta; and several other amyloidogenic proteins likely recognizing a potentially common amyloid structural aptatope21. Here, we present the SELEX methodology used in production of these aptamers21.

Selection of Aptamers for Amyloid &#946;-Protein, the Causative Agent of Alzheimer's Disease

Aptamers are short ribo-/deoxyribo-oligonucleotides selected by in-vitro evolution methods based on affinity for a specific target. Aptamers are molecular recognition tools with versatile therapeutic, diagnostic, and research applications. We demonstrate methods for selection of aptamers for amyloid &beta;-protein, the causative agent of Alzheimer's disease.

اختيار الأبتامرات للبروتين اميلويد - β ، والمسبب لمرض الزهايمر

Alzheimer's disease (AD) is a progressive, age-dependent, neurodegenerative disorder with an insidious course that renders its presymptomatic diagnosis ...

Neuroscience

A Tailored HPLC Purification Protocol That Yields High-purity Amyloid Beta 42 and Amyloid Beta 40 Peptides, Capable of Oligomer Formation

Amyloidogenic peptides such as the Alzheimer's disease-implicated Amyloid beta (A&#946;), can present a significant challenge when trying to obtain high purity material. Here we present a tailored HPLC purification protocol to produce high-purity amyloid beta 42 (A&#946;42) and amyloid beta 40 (A&#946;40) peptides. We have found that the combination of commercially available hydrophobic poly(styrene/divinylbenzene) stationary phase, polymer laboratory reverse phase - styrenedivinylbenzene (PLRP-S) under high pH conditions, enables the attainment of high purity (&gt;95%) A&#946;42 in a single chromatographic run. The purification is highly reproducible and can be amended to both semi-preparative and analytical conditions depending upon the amount of material wished to be purified. The protocol can also be applied to the A&#946;40 peptide with identical success and without the need to alter the method.

Herein we report a tailored HPLC purification protocol that yields high-purity amyloid beta 42 (A&#946;42) and amyloid beta 40 (A&#946;40) peptides, capable of oligomer formation. Amyloid beta is a highly aggregation prone, hydrophobic peptide implicated in Alzheimer&#39;s disease. The amyloidogenic nature of the peptide makes its purification a challenge.

بروتوكول تنقية مصممة HPLC أن ينتج عالية النقاء اميلويد بيتا اميلويد بيتا 42 و 40 الببتيدات، قادرة على قليل وحدات تشكيل

Amyloidogenic peptides such as the Alzheimer's disease-implicated Amyloid beta (A&#946;), can present a significant challenge when trying to obtain high ...

Biochemistry

Method for Efficient Refolding and Purification of Chemoreceptor Ligand Binding Domain

Identification of natural ligands of chemoreceptors and structural studies aimed at elucidation of the molecular basis of the ligand specificity can be greatly facilitated by the production of milligram amounts of pure, folded ligand binding domains. Attempts to heterologously express periplasmic ligand binding domains of bacterial chemoreceptors in Escherichia coli (E. coli) often result in their targeting into inclusion bodies. Here, a method is presented for protein recovery from inclusion bodies, its refolding and purification, using the periplasmic dCACHE ligand binding domain of Campylobacter jejuni (C. jejuni) chemoreceptor Tlp3 as an example. The approach involves expression of the protein of interest with a cleavable His6-tag, isolation and urea-mediated solubilisation of inclusion bodies, protein refolding by urea depletion, and purification by means of affinity chromatography, followed by tag removal and size-exclusion chromatography. The circular dichroism spectroscopy is used to confirm the folded state of the pure protein. It has been demonstrated that this protocol is generally useful for production of milligram amounts of dCACHE periplasmic ligand binding domains of other bacterial chemoreceptors in a soluble and crystallisable form.

A procedure is presented for the refolding of the dCACHE periplasmic ligand binding domain of Campylobacter jejuni chemoreceptor Tlp3 from inclusion bodies and the purification to yield milligram quantities of protein.

الأسلوب كفاءة ريفولدينج وتنقية ليجند تشيموريسيبتور ملزمة المجال

Identification of natural ligands of chemoreceptors and structural studies aimed at elucidation of the molecular basis of the ligand specificity can be ...

Screening for Amyloid Aggregation by Semi-Denaturing Detergent-Agarose Gel Electrophoresis

Amyloid aggregation is associated with numerous protein misfolding pathologies and underlies the infectious properties of prions, which are conformationally self-templating proteins that are thought to have beneficial roles in lower organisms.  Amyloids have been notoriously difficult to study due to their insolubility and structural heterogeneity.  However, resolution of amyloid polymers based on size and detergent insolubility has been made possible by Semi-Denaturing Detergent-Agarose Gel Electrophoresis (SDD-AGE).  This technique is finding widespread use for the detection and characterization of amyloid conformational variants.  Here, we demonstrate an adaptation of this technique that facilitates its use in large-scale applications, such as screens for novel prions and other amyloidogenic proteins.  The new SDD-AGE method uses capillary transfer for greater reliability and ease of use, and allows any sized gel to be accomodated.  Thus, a large number of samples, prepared from cells or purified proteins, can be processed simultaneously for the presence of SDS-insoluble conformers of tagged proteins.

SDD-AGE is a useful technique for the detection and characterization of amyloid-like polymers in cells. Here we demonstrate an adaptation that makes this technique amenable to large-scale applications.

الكشف عن تجميع اميلويد بواسطة تغيير طبيعة شبه الكهربائي جل منظفات - Agarose

Amyloid aggregation is associated with numerous protein misfolding pathologies and underlies the infectious properties of prions, which are ...

Purification and Aggregation of the Amyloid Precursor Protein Intracellular Domain

Amyloid precursor protein (APP) is a type I transmembrane protein associated with the pathogenesis of Alzheimer's disease (AD). APP is characterized by a large extracellular domain and a short cytosolic domain termed the APP intracellular domain (AICD). During maturation through the secretory pathway, APP can be cleaved by proteases termed &alpha;, &beta;, and &gamma;-secretases1. Sequential proteolytic cleavage of APP with &beta; and &gamma;-secretases leads to the production of a small proteolytic peptide, termed A&beta;, which is amyloidogenic and the core constituent of senile plaques. The AICD is also liberated from the membrane after secretase processing, and through interactions with Fe65 and Tip60, can translocate to the nucleus to participate in transcription regulation of multiple target genes2,3. Protein-protein interactions involving the AICD may affect trafficking, processing, and cellular functions of holo-APP and its C-terminal fragments. We have recently shown that AICD can aggregate in vitro, and this process is inhibited by the AD-implicated molecular chaperone ubiquilin-14. Consistent with these findings, the AICD has exposed hydrophobic domains and is intrinsically disordered in vitro5,6, however it obtains stable secondary structure when bound to Fe657. We have proposed that ubiquilin-1 prevents inappropriate inter- and intramolecular interactions of AICD, preventing aggregation in vitro and in intact cells4. While most studies focus on the role of APP in the pathogenesis of AD, the role of AICD in this process is not clear. Expression of AICD has been shown to induce apoptosis8, to modulate signaling pathways9, and to regulate calcium signaling10. Over-expression of AICD and Fe65 in a transgenic mouse model induces Alzheimer's like pathology11, and recently AICD has been detected in brain lysates by western blotting when using appropriate antigen retrieval techniques12. To facilitate structural, biochemical, and biophysical studies of the AICD, we have developed a procedure to produce recombinantly large amounts of highly pure AICD protein. We further describe a method for inducing the in vitro thermal aggregation of AICD and analysis by atomic force microscopy. The methods described are useful for biochemical, biophysical, and structural characterization of the AICD and the effects of molecular chaperones on AICD aggregation.

A method for large-scale purification of the APP intracellular domain (AICD) is described. We also describe methodology to induce in vitro AICD aggregation and visualization by atomic force microscopy. The methods described are useful for biochemical/structural characterization of the AICD and the effects of molecular chaperones on its aggregation.

تنقية وتجميع على البروتين اميلويد السلائف المجال بين الخلايا

Amyloid precursor protein (APP) is a type I transmembrane protein associated with the pathogenesis of Alzheimer's disease (AD). APP is characterized by a ...

Medicine

Expression, Purification, Crystallization, and Enzyme Assays of Fumarylacetoacetate Hydrolase Domain-Containing Proteins

Fumarylacetoacetate hydrolase (FAH) domain-containing proteins (FAHD) are identified members of the FAH superfamily in eukaryotes. Enzymes of this superfamily generally display multi-functionality, involving mainly hydrolase and decarboxylase mechanisms. This article presents a series of consecutive methods for the expression and purification of FAHD proteins, mainly FAHD protein 1 (FAHD1) orthologues among species (human, mouse, nematodes, plants, etc.). Covered methods are protein expression in E. coli, affinity chromatography, ion exchange chromatography, preparative and analytical gel filtration, crystallization, X-ray diffraction, and photometric assays. Concentrated protein of high levels of purity (&#62;98%) may be employed for crystallization or antibody production. Proteins of similar or lower quality may be employed in enzyme assays or used as antigens in detection systems (Western-Blot, ELISA). In the discussion of this work, the identified enzymatic mechanisms of FAHD1 are outlined to describe its hydrolase and decarboxylase bi-functionality in more detail.

Expression and purification of fumarylacetoacetate hydrolase domain-containing proteins is described with examples (expression in E. coli, FPLC). Purified proteins are used for crystallization and antibody production and employed for enzyme assays. Selected photometric assays are presented to display the multi-functionality of FAHD1 as oxaloacetate decarboxylase and acylpyruvate hydrolase.

التعبير، تنقية، تبلور، واختبارات إنزيم من البروتينات التي تحتوي على مجال الهيدرولاز اتاتس

Fumarylacetoacetate hydrolase (FAH) domain-containing proteins (FAHD) are identified members of the FAH superfamily in eukaryotes. Enzymes of this ...

Purification of Hsp104, a Protein Disaggregase

Hsp104 is a hexameric AAA+ protein1 from yeast, which couples ATP hydrolysis to protein disaggregation2-10 (Fig. 1). This activity imparts two key selective advantages. First, renaturation of disordered aggregates by Hsp104 empowers yeast survival after various protein-misfolding stresses, including heat shock3,5,11,12. Second, remodeling of cross-beta amyloid fibrils by Hsp104 enables yeast to exploit myriad prions (infectious amyloids) as a reservoir of beneficial and heritable phenotypic variation13-22. Remarkably, Hsp104 directly remodels preamyloid oligomers and amyloid fibrils, including those comprised of the yeast prion proteins Sup35 and Ure223-30. This amyloid-remodeling functionality is a specialized facet of yeast Hsp104. The E. coli orthologue, ClpB, fails to remodel preamyloid oligomers or amyloid fibrils26,31,32.
Hsp104 orthologues are found in all kingdoms of life except, perplexingly, animals. Indeed, whether animal cells possess any enzymatic system that couples protein disaggregation to renaturation (rather than degradation) remains unknown33-35. Thus, we and others have proposed that Hsp104 might be developed as a therapeutic agent for various neurodegenerative diseases connected with the misfolding of specific proteins into toxic preamyloid oligomers and amyloid fibrils4,7,23,36-38. There are no treatments that directly target the aggregated species associated with these diseases. Yet, Hsp104 dissolves toxic oligomers and amyloid fibrils composed of alpha-synuclein, which are connected with Parkinson's Disease23 as well as amyloid forms of PrP39. Importantly, Hsp104 reduces protein aggregation and ameliorates neurodegeneration in rodent models of Parkinson's Disease23 and Huntington's disease38. Ideally, to optimize therapy and minimize side effects, Hsp104 would be engineered and potentiated to selectively remodel specific aggregates central to the disease in question4,7. However, the limited structural and mechanistic understanding of how Hsp104 disaggregates such a diverse repertoire of aggregated structures and unrelated proteins frustrates these endeavors30,40-42.
To understand the structure and mechanism of Hsp104, it is essential to study the pure protein and reconstitute its disaggregase activity with minimal components. Hsp104 is a 102kDa protein with a pI of ~5.3, which hexamerizes in the presence of ADP or ATP, or at high protein concentrations in the absence of nucleotide43-46. Here, we describe an optimized protocol for the purification of highly active, stable Hsp104 from E. coli. The use of E. coli allows simplified large-scale production and our method can be performed quickly and reliably for numerous Hsp104 variants. Our protocol increases Hsp104 purity and simplifies His6-tag removal compared to a previous purification method from E. coli47. Moreover, our protocol is more facile and convenient than two more recent protocols26,48.

Here, we describe a protocol for the purification of highly active Hsp104, a hexameric AAA+ protein from yeast, which couples ATP hydrolysis to protein disaggregation. This scheme exploits a His6-tagged construct for affinity purification from E. coli followed by anion-exchange chromatography, His6-tag removal with TEV protease, and size-exclusion chromatography.

تنقية Hsp104 ، وهو بروتين Disaggregase

Hsp104 is a hexameric AAA+ protein1 from yeast, which couples ATP hydrolysis to protein disaggregation2-10 (Fig. 1). This activity imparts two key ...

Rapid Generation of Amyloid from Native Proteins In vitro

Proteins carry out crucial tasks in organisms by exerting functions elicited from their specific three dimensional folds. Although the native structures of polypeptides fulfill many purposes, it is now recognized that most proteins can adopt an alternative assembly of beta-sheet rich amyloid. Insoluble amyloid fibrils are initially associated with multiple human ailments, but they are increasingly shown as functional players participating in various important cellular processes. In addition, amyloid deposited in patient tissues contains nonproteinaceous components, such as nucleic acids and glycosaminoglycans (GAGs). These cofactors can facilitate the formation of amyloid, resulting in the generation of different types of insoluble precipitates. By taking advantage of our understanding how proteins misfold via an intermediate stage of soluble amyloid precursor, we have devised a method to convert native proteins to amyloid fibrils in vitro. This approach allows one to prepare amyloid in large quantities, examine the properties of amyloid generated from specific proteins, and evaluate the structural changes accompanying the conversion.

Rapid Generation of Amyloid from Native Proteins In vitro

Proteins can either adopt a native structure or misfold into insoluble amyloid. Conditions that favor the misfolding pathway lead to the formation of different types of amyloid fibrils. The methods described here allow rapid conversion of native proteins into amyloid in vitro.

Proteins carry out crucial tasks in organisms by exerting functions elicited from their specific three dimensional folds. Although the native structures ...

Biochemical Purification and Proteomic Characterization of Amyloid Fibril Cores from the Brain

Proteinaceous fibrillar inclusions are key pathological hallmarks of multiple neurodegenerative diseases. In the early stages of Alzheimer&#39;s disease (AD), amyloid-beta peptides form protofibrils in the extracellular space, which act as seeds that gradually grow and mature into large amyloid plaques. Despite this basic understanding, current knowledge of the amyloid fibril structure, composition, and deposition patterns in the brain is limited. One major barrier has been the inability to isolate highly purified amyloid fibrils from brain extracts. Affinity purification and laser capture microdissection-based approaches have been previously used to isolate amyloids but are limited by the small quantity of material that can be recovered. This novel, robust protocol describes the biochemical purification of amyloid plaque cores using sodium dodecyl sulfate (SDS) solubilization with sucrose density gradient ultracentrifugation and ultrasonication and yields highly pure fibrils from AD patients and AD model brain tissues. Mass spectrometry (MS)-based bottom-up proteomic analysis of the purified material represents a robust strategy to identify nearly all the primary protein components of amyloid fibrils. Previous proteomic studies of proteins in the amyloid coronae have revealed an unexpectedly large and functionally diverse collection of proteins. Notably, after refining the purification strategy, the number of co-purifying proteins was reduced by more than 10-fold, indicating the high purity of the isolated SDS insoluble material. Negative staining and immuno-gold electron microscopy allowed confirmation of the purity of these preparations. Further studies are required to understand the spatial and biological attributes that contribute to the deposition of these proteins into amyloid inclusions. Taken together, this analytical strategy is well-positioned to increase the understanding of amyloid biology.

This biochemical purification method with mass spectrometry-based proteomic analysis facilitates the robust characterization of amyloid fibril cores, which may accelerate the identification of targets for preventing Alzheimer's disease.

التنقية البيوكيميائية والتوصيف البروتيني للنوى الليفية الأميلويد من الدماغ

Proteinaceous fibrillar inclusions are key pathological hallmarks of multiple neurodegenerative diseases. In the early stages of Alzheimer&#39;s disease ...

Isolation of Amyloid Fibrils from Brain Tissue Extract

Source: Upadhyay, A. et al., <a href="https://app.jove.com/v/63816">Biochemical Purification and Proteomic Characterization of Amyloid Fibril Cores from the Brain.</a> J. Vis. Exp. (2022)
This video demonstrates the protocol for isolating&#160; amyloid fibrils from mouse brain tissue extract using density gradient centrifugation and digestion of non-amyloid proteins.

عزل ألياف الأميلويد من مستخلص أنسجة المخ

All procedures involving animal samples have been reviewed and approved by the appropriate animal ethical review committee.
1. Tissue harvesting and ...

تنقية وRefolding إلى اميلويد الألياف من (صاحب)

Summary

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اختيار الأبتامرات للبروتين اميلويد - β ، والمسبب لمرض الزهايمر

بروتوكول تنقية مصممة HPLC أن ينتج عالية النقاء اميلويد بيتا اميلويد بيتا 42 و 40 الببتيدات، قادرة على قليل وحدات تشكيل

الأسلوب كفاءة ريفولدينج وتنقية ليجند تشيموريسيبتور ملزمة المجال

الكشف عن تجميع اميلويد بواسطة تغيير طبيعة شبه الكهربائي جل منظفات - Agarose

تنقية وتجميع على البروتين اميلويد السلائف المجال بين الخلايا

التعبير، تنقية، تبلور، واختبارات إنزيم من البروتينات التي تحتوي على مجال الهيدرولاز اتاتس

تنقية Hsp104 ، وهو بروتين Disaggregase

Rapid Generation of Amyloid from Native Proteins In vitro

التنقية البيوكيميائية والتوصيف البروتيني للنوى الليفية الأميلويد من الدماغ

عزل ألياف الأميلويد من مستخلص أنسجة المخ