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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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 ...

purification-refolding-to-amyloid-fibrils-his-6-tagged-recombinant

Research

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Biology

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

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 ...

Biochemistry

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

Synthesis of an Intein-mediated Artificial Protein Hydrogel

We present the synthesis of a highly stable protein hydrogel mediated by a split-intein-catalyzed protein trans-splicing reaction. The building blocks of this hydrogel are two protein block-copolymers each containing a subunit of a trimeric protein that serves as a crosslinker and one half of a split intein. A highly hydrophilic random coil is inserted into one of the block-copolymers for water retention. Mixing of the two protein block copolymers triggers an intein trans-splicing reaction, yielding a polypeptide unit with crosslinkers at either end that rapidly self-assembles into a hydrogel. This hydrogel is very stable under both acidic and basic conditions, at temperatures up to 50 &#176;C, and in organic solvents. The hydrogel rapidly reforms after shear-induced rupture. Incorporation of a &#34;docking station peptide&#34; into the hydrogel building block enables convenient incorporation of &#34;docking protein&#34;-tagged target proteins. The hydrogel is compatible with tissue culture growth media, supports the diffusion of 20 kDa molecules, and enables the immobilization of bioactive globular proteins. The application of the intein-mediated protein hydrogel as an organic-solvent-compatible biocatalyst was demonstrated by encapsulating the horseradish peroxidase enzyme and corroborating its activity.

We present the synthesis of a split-intein-mediated protein hydrogel. The building blocks of this hydrogel are two protein copolymers each containing a subunit of a trimeric protein that serves as a crosslinker and one half of a split intein. Mixing of the two protein copolymers triggers an intein trans-splicing reaction, yielding a polypeptide unit that self-assembles into a hydrogel. This hydrogel is highly pH- and temperature-stable, compatible with organic solvents, and easily incorporates functional globular proteins.

التوليف من البروتين الاصطناعي Intein بوساطة هيدروجيل

We present the synthesis of a highly stable protein hydrogel mediated by a split-intein-catalyzed protein trans-splicing reaction. The building blocks of ...

Bioengineering

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 ...

Targeting Cysteine Thiols for in Vitro Site-specific Glycosylation of Recombinant Proteins

Stromal interaction molecule-1 (STIM1) is a type-I transmembrane protein located on the endoplasmic reticulum (ER) and plasma membranes (PM). ER-resident STIM1 regulates the activity of PM Orai1 channels in a process known as store operated calcium (Ca2+) entry which is the principal Ca2+ signaling process that drives the immune response. STIM1 undergoes post-translational N-glycosylation at two luminal Asn sites within the Ca2+ sensing domain of the molecule. However, the biochemical, biophysical, and structure biological effects of N-glycosylated STIM1 were poorly understood until recently due to an inability to readily obtain high levels of homogeneous N-glycosylated protein. Here, we describe the implementation of an in vitro chemical approach which attaches glucose moieties to specific protein sites applicable to understanding the underlying effects of N-glycosylation on protein structure and mechanism. Using solution nuclear magnetic resonance spectroscopy we assess both efficiency of the modification as well as the structural consequences of the glucose attachment with a single sample. This approach can readily be adapted to study the myriad glycosylated proteins found in nature.

Targeting Cysteine Thiols for in Vitro Site-specific Glycosylation of Recombinant Proteins

Biochemical and structural analyses of glycosylated proteins require relatively large amounts of homogeneous samples. Here, we present an efficient chemical method for site-specific glycosylation of recombinant proteins purified from bacteria by targeting reactive Cys thiols.

استهداف ثيولس سيستين في المختبر جليكوسيليشن الخاصة بالموقع من البروتينات المؤتلف

Stromal interaction molecule-1 (STIM1) is a type-I transmembrane protein located on the endoplasmic reticulum (ER) and plasma membranes (PM). ER-resident ...

Immunology and Infection

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 ...

Production of E. coli-expressed Self-Assembling Protein Nanoparticles for Vaccines Requiring Trimeric Epitope Presentation

Self-assembling protein nanoparticles (SAPNs) function as repetitive antigen displays and can be used to develop a wide range of vaccines for different infectious diseases. In this article we demonstrate a method to produce a SAPN core containing a six-helix bundle (SHB) assembly that is capable of presenting antigens in a trimeric conformation. We describe the expression of the SHB-SAPN in an E. coli system, as well as the necessary protein purification steps. We included an isopropanol wash step to reduce the residual bacterial lipopolysaccharide. As an indication of the protein identity and purity, the protein reacted with known monoclonal antibodies in Western blot analyses. After refolding, the size of the particles fell in the expected range (20 to 100 nm), which was confirmed by dynamic light scattering, nanoparticle tracking analysis, and transmission electron microscopy. The methodology described here is optimized for the SHB-SAPN, however, with only slight modifications it can be applied to other SAPN constructs. This method is also easily transferable to large scale production for GMP manufacturing for human vaccines.

Production of E. coli-expressed Self-Assembling Protein Nanoparticles for Vaccines Requiring Trimeric Epitope Presentation

A detailed method is provided here describing the purification, refolding, and characterization of self-assembling protein nanoparticles (SAPNs) for use in vaccine development.

إنتاج E.القولونية - أعرب الذاتي تجميع البروتين الجسيمات النانوية لللقاحات التي تتطلب عرض الإبإبيلة الانتهازية

Self-assembling protein nanoparticles (SAPNs) function as repetitive antigen displays and can be used to develop a wide range of vaccines for different ...

A Purification and In Vitro Activity Assay for a (p)ppGpp Synthetase from Clostridium difficile

Kinase and pyrophosphokinase enzymes transfer the gamma phosphate or the beta-gamma pyrophosphate moiety from nucleotide triphosphate precursors to substrates to create phosphorylated products. The use of &#947;-32-P labeled NTP precursors allows simultaneous monitoring of substrate utilization and product formation by radiography. Thin layer chromatography (TLC) on cellulose plates allows rapid separation and sensitive quantification of substrate and product. We present a method for utilizing the thin-layer chromatography to assay the pyrophosphokinase activity of a purified (p)ppGpp synthetase. This method has previously been used to characterize the activity of cyclic nucleotide and dinucleotide synthetases and is broadly suitable for characterizing the activity of any enzyme that hydrolyzes a nucleotide triphosphate bond or transfers a terminal phosphate from a phosphate donor to another molecule.

A Purification and In Vitro Activity Assay for a pppGpp Synthetase from Clostridium difficile

Here, we describe a method for purifying histidine-tagged pyrophosphokinase enzymes and utilizing thin layer chromatography of radiolabelled substrates and products to assay for the enzymatic activity in vitro. The enzyme activity assay is broadly applicable to any kinase, nucleotide cyclase, or phosphor-transfer reaction whose mechanism includes nucleotide triphosphate hydrolysis.

تنقية و في المختبر نشاط الإنزيم (ع) بجب سينثاتيز من المطثيّة العسيرة

Kinase and pyrophosphokinase enzymes transfer the gamma phosphate or the beta-gamma pyrophosphate moiety from nucleotide triphosphate precursors to ...

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 ...

تنقية وRefolding إلى اميلويد الألياف من (صاحب)

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الأسلوب كفاءة ريفولدينج وتنقية ليجند تشيموريسيبتور ملزمة المجال

تنقية وتجميع على البروتين اميلويد السلائف المجال بين الخلايا

التوليف من البروتين الاصطناعي Intein بوساطة هيدروجيل

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

استهداف ثيولس سيستين في المختبر جليكوسيليشن الخاصة بالموقع من البروتينات المؤتلف

تنقية Hsp104 ، وهو بروتين Disaggregase

إنتاج E.القولونية - أعرب الذاتي تجميع البروتين الجسيمات النانوية لللقاحات التي تتطلب عرض الإبإبيلة الانتهازية

تنقية و في المختبر نشاط الإنزيم (ع) بجب سينثاتيز من المطثيّة العسيرة

Rapid Generation of Amyloid from Native Proteins In vitro

التنقية البيوكيميائية والتوصيف البروتيني للنوى الليفية الأميلويد من الدماغ