Name: purification of hsp104, a protein disaggregase
Uploaded: 2011-09-30T13:00:00
Description: Watch this Scientific Journal Video about Purification of Hsp104, a Protein Disaggregase at JoVE.com

यह काम (5T32GM008275 22) NIH और एक अमेरिकन हार्ट एसोसिएशन predoctoral फेलोशिप (ईएएस के लिए) से एक अनुदान द्वारा समर्थित किया गया, रसायन विज्ञान जीवविज्ञान (2T32GM071339 06A1) NIH (मेड करने के लिए) से इंटरफ़ेस फैलोशिप, और से अनुदान (1DP2OD002177-01 और NS067354 - 0,110) एनआईएच, एलिसन मेडिकल फाउंडेशन, और बिल और मेलिंडा गेट्स फाउंडेशन (जे एस के लिए).

1. Hsp104 की अभिव्यक्ति <ol><li> प्लाज्मिड ई. में शुद्धि के लिए नियोजित कोलाई, pPROEX HTB-Hsp104 Hsp104 TRC 26 प्रमोटर के inducible नियंत्रण के तहत खुले पढ़ने फ्रेम शामिल हैं . प्लाज्मिड एन टर्मिनल उनकी 6 टैग कि TEV protease दरा?...

समयरेखा: हम अधिक से अधिक Hsp104 गतिविधि के लिए सुझाव है कि पूरे शुद्धि योजना के रूप में तेजी से संभव के रूप में पूरा किया जाना. हालांकि, शुद्धि चरणों की संख्या एक मांग अनुसूची है कि हमेशा व्यावहारिक नहीं ह?...

<table border="1"><tbody><tr><th> अभिकर्मक के नाम </th><th> कंपनी </th><th> सूचीपत्र संख्या </th></tr><tr><td> BL21 - CodonPlus - आरआईएल सक्षम कक्ष </td><td> Stratagene, Agilent टेक्नोलॉजीज </td><td> 230255 </td></tr><tr><td> 2XYT शोरबा </td><td> यूएसबी </td><td> 75864 </td></tr><tr><td> पूर्ण, मिनी, EDTA मुक्त protease अवरोध करनेवाला गोलियाँ </td><td> रॉश </td><td> 1836170 </td></tr><tr><td> एक Pepstatin </td><td> सिग्मा </td><td> P4265 </td></tr><tr><td> नी Sepharose 6 फास्ट फ्लो </td><td> जीई हेल्थकेयर </td><td> 17-5318-02 </td></tr><tr><td> Amicon - अल्ट्रा 15 केन्द्रापसारक फ़िल्टर इकाइयों (तीस हज़ार MWCO) </td><td> Millipore </td><td> UFC903008 </td></tr><tr><td> संसाधन क्यू 6ml स्तंभ - </td><td> जीई हेल्थकेयर </td><td> 17-1179-01 </td></tr><tr><td> proTEV Protease </td><td> Promega </td><td> V6052 </td></tr><tr><td> AcTEV Protease </td><td> Invitrogen </td><td> 12575015 </td></tr><tr><td> Superose 6 10/300 जीएल </td><td> जीई हेल्थकेयर </td><td> 17-5172-01 </td></tr><tr><td> Hsp40 </td><td> परख डिजाइन </td><td> SPP-400 </td></tr><tr><td> Hsp72 </td><td> परख डिजाइन </td><td> ADI - NSP-555 </td></tr></tbody></table>

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.

Watch this Scientific Journal Video about Purification of Hsp104, a Protein Disaggregase at JoVE.com

Purification of Hsp104, a Protein Disaggregase

यहाँ, हम अत्यधिक सक्रिय Hsp104, hexameric + खमीर, जो जोड़ों एटीपी प्रोटीन disaggregation hydrolysis से एएए प्रोटीन की शुद्धि के लिए एक प्रोटोकॉल का वर्णन. इस योजना के कारनामे His6 टैग से आत्मीयता शुद्धि के लिए का निर्माण<em&gt; ई. कोलाई</em&gt; आयनों मुद्रा क्रोमैटोग्राफी, TEV protease के साथ His6 टैग हटाने, और आकार अपवर्जन क्रोमैटोग्राफी द्वारा पीछा किया.

Hsp104 के शोधन, एक प्रोटीन Disaggregase

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

Research

JoVE Journal

Biology

Staining of Proteins in Gels with Coomassie G-250 without Organic Solvent and Acetic Acid

कार्बनिक सॉल्वेंट और एसिटिक एसिड के बिना जी 250 Coomassie के साथ जैल में प्रोटीन की धुंधला

In classical protein staining protocols using Coomassie Brilliant Blue (CBB), solutions with high contents of toxic and flammable organic solvents ...

Purification of the M. magneticum Strain AMB-1 Magnetosome Associated Protein MamA&Delta;41

Purification of the M. magneticum Strain AMB-1 Magnetosome Associated Protein MamAÃƒÅ½Ã¢â‚¬Â41

के शोधन एम. magneticum तनाव Magnetosome एसोसिएटेड प्रोटीन MamAΔ41-1 AMB

Magnetotactic bacteria comprise a diverse group of aquatic microorganisms that are able to orientate themselves along geomagnetic fields. This behavior is ...

Expression, Detergent Solubilization, and Purification of a Membrane Transporter, the MexB Multidrug Resistance Protein

अभिव्यक्ति, डिटर्जेंट solubilization, और एक झिल्ली ट्रांसपोर्टर, MexB बहुऔषध प्रतिरोध प्रोटीन के शोधन

Multidrug resistance (MDR), the ability of a cancer cell or pathogen to be resistant to a wide range of structurally and functionally unrelated ...

Automated Hydrophobic Interaction Chromatography Column Selection for Use in Protein Purification

स्वचालित hydrophobic प्रोटीन शुद्धीकरण में प्रयोग के लिए सहभागिता क्रोमैटोग्राफी स्तंभ चयन

In contrast to other chromatographic methods for purifying proteins (e.g. gel filtration, affinity, and ion exchange), hydrophobic interaction ...

Chromatographic Purification of Highly Active Yeast Ribosomes

अति सक्रिय खमीर Ribosomes के chromatographic शोधन

Eukaryotic ribosomes are much more labile as compared to their eubacterial and archael counterparts, thus posing a significant challenge to researchers.  ...

Orthogonal Protein Purification Facilitated by a Small Bispecific Affinity Tag

विषयेतर प्रोटीन एक लघु bispecific आत्मीयता टैग द्वारा सुविधा शोधन

Due to the high costs associated with purification of recombinant proteins the protocols need to be rationalized. For high-throughput efforts there is a ...

Identification of Protein Interacting Partners Using Tandem Affinity Purification

प्रोटीन बातचीत मिलकर आत्मीयता शोधन का उपयोग पार्टनर्स की पहचान

A critical and often limiting step in understanding the function of host and viral proteins is the identification of interacting cellular or viral protein ...

Generation, Purification, and Characterization of Cell-invasive DISC1 Protein Species

जनरेशन, शोधन, सेल इनवेसिव DISC1 प्रोटीन प्रजाति की विशेषता और

Protein aggregation is seen as a general hallmark of chronic, degenerative brain conditions like, for example, in the neurodegenerative diseases ...

Protein Purification-free Method of Binding Affinity Determination by Microscale Thermophoresis

Microscale Thermophoresis द्वारा बंधन आत्मीयता निर्धारण के प्रोटीन शुद्धीकरण मुक्त विधि

Quantitative characterization of protein  interactions is essential in practically any field of life sciences,  particularly drug discovery. Most of ...

Identifying Protein-protein Interaction in Drosophila Adult Heads by Tandem Affinity Purification (TAP)

Identifying Protein-protein Interaction in Drosophila Adult Heads by Tandem Affinity Purification TAP

में प्रोटीन, प्रोटीन बातचीत की पहचान<em&gt; ड्रोसोफिला</emअग्रानुक्रम आत्मीयता शुद्धि द्वारा&gt; वयस्क प्रमुखों (नल)

Genetic screens conducted using Drosophila melanogaster (fruit fly) have made numerous milestone discoveries in the advance of biological sciences. ...