Name: high-throughput purification of affinity-tagged recombinant proteins
Uploaded: 2012-08-26T14:00:00
Duration: 7 min 44 s
Description: Watch this Scientific Journal Video about High-throughput Purification of Affinity-tagged Recombinant Proteins at JoVE.com

यह काम एक वेलकम परियोजना अनुदान (078043/Z/05/Z) द्वारा ROJW समर्थित किया गया

<ol>
	<li>Weinzierl, R. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+nucleotide+addition+cycle+of+RNA+polymerase+is+controlled+by+two+molecular+hinges+in+the+Bridge+Helix+domain.">The nucleotide addition cycle of RNA polymerase is controlled by two molecular hinges in the Bridge Helix domain.</a> BMC Biol. 8, 1741-7007 (2010).</li><li>Nottebaum, S., Tan, L., Trzaska, D., Carney, H. C., Weinzierl, R. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+RNA+polymerase+factory:+a+robotic+in+vitro+assembly+platform+for+high-throughput+production+of+recombinant+protein+complexes.">The RNA polymerase factory: a robotic in vitro assembly platform for high-throughput production of recombinant protein complexes.</a> Nucleic Acids Res. 36, 245-252 (2008).</li><li>Tan, L., Wiesler, S., Trzaska, D., Carney, H. C., Weinzierl, R. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bridge+helix+and+trigger+loop+perturbations+generate+superactive+RNA+polymerases.">Bridge helix and trigger loop perturbations generate superactive RNA polymerases.</a> J. Biol. 7, 40 (2008).</li><li>Wiesler, S. C., Weinzierl, R. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+linker+domain+of+basal+transcription+factor+TFIIB+controls+distinct+recruitment+and+transcription+stimulation+functions.">The linker domain of basal transcription factor TFIIB controls distinct recruitment and transcription stimulation functions.</a> Nucleic Acids Res. 39, 464-474 (2011).</li><li>Weinzierl, R. O., Wiesler, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Revealing+the+Function+of+TFIIB.">Revealing the Function of TFIIB.</a> Transcription. , (2011).</li><li>Bell, S. D., Magill, C. P., Jackson, S. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Basal+and+regulated+transcription+in+Archaea.">Basal and regulated transcription in Archaea.</a> Biochemical Society transactions. 29, 392-395 (2001).</li><li>Parvin, J. D., Sharp, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+topology+and+a+minimal+set+of+basal+factors+for+transcription+by+RNA+polymerase+II.">DNA topology and a minimal set of basal factors for transcription by RNA polymerase II.</a> Cell. 73, 533-540 (1993).</li><li>Tyree, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+a+minimal+set+of+proteins+that+is+sufficient+for+accurate+initiation+of+transcription+by+RNA+polymerase+II.">Identification of a minimal set of proteins that is sufficient for accurate initiation of transcription by RNA polymerase II.</a> Genes. 7, 1254-1265 (1993).</li><li>Batada, N. N., Westover, K. D., Bushnell, D. A., Levitt, M., Kornberg, R. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diffusion+of+nucleoside+triphosphates+and+role+of+the+entry+site+to+the+RNA+polymerase+II+active+center.">Diffusion of nucleoside triphosphates and role of the entry site to the RNA polymerase II active center.</a> Proceedings of the National Academy of Sciences of the United States of America. 101, 17361-17364 (2004).</li><li>Liu, X., Bushnell, D. A., Wang, D., Calero, G., Kornberg, R. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structure+of+an+RNA+Polymerase+II-TFIIB+Complex+and+the+Transcription+Initiation+Mechanism.">Structure of an RNA Polymerase II-TFIIB Complex and the Transcription Initiation Mechanism.</a> Science (New York, N.Y). 327, 206-209 (2010).</li><li>Kostrewa, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNA+polymerase+II-TFIIB+structure+and+mechanism+of+transcription+initiation.">RNA polymerase II-TFIIB structure and mechanism of transcription initiation.</a> Nature. 462, 323-330 (2010).</li><li>Werner, F., Weinzierl, R. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+modulation+of+RNA+polymerase+core+functions+by+basal+transcription+factors.">Direct modulation of RNA polymerase core functions by basal transcription factors.</a> Molecular and cellular biology. 25, 8344-8355 (2005).</li></ol>

ब्याज की कोई संघर्ष की घोषणा की.

स्वचालित पुनः संयोजक प्रोटीन शोधन विधि यहाँ वर्णित उत्परिवर्ती प्रोटीन की एक बड़ी संख्या के उत्पादन और शोधन के एक छोटे पैमाने पर प्रारूप में न्यूनतम मानवीय हस्तक्षेप के साथ अत्यधिक प्रतिलिपि प्रस्तुत करने की शर्तों के तहत आंकड़े 1 और 2 व्यवस्थित गुणवत्ता नियंत्रण और के उदाहरण के शो के परिणाम की अनुमति देता है. शुट्ठ प्रोटीन चित्रा 3 से पता चलता है कि शुद्ध प्रतिलेखन कारकों इस उदाहरण में प्रयुक्त कार्यात्मक assays में एक अत्यधिक प्रतिलिपि प्रस्तुत करने योग्य तरीके में प्रदर्शन. हालांकि प्रक्रिया archaeal TFIIB की शुद्धि के लिए विकसित किया गया था, यह आत्मीयता टैग प्रोटीन की शुद्धि के लिए व्यापक रूप से लागू होता है. ऐसे स्वचालित शोधन प्रोटोकॉल का उपयोग इस प्रकार पुनः संयोजक प्रोटीन की जैव रासायनिक विश्लेषण काफी सुविधा होगी और इस तरह से है कि मैन्युअल रूप से प्राप्त करने के लिए मुश्किल है पैमाने पर प्रोटीन, प्रोटीन बातचीत की हमारी समझ को आगे जाएगा.

<table border="1"><tbody><tr><td> अभिकर्मक के नाम </td><td> कंपनी </td><td> सूचीपत्र संख्या </td><td> टिप्पणियां (वैकल्पिक) </td></tr><tr><td> ओवरनाइट एक्सप्रेस त्वरित टीबी मध्यम </td><td> मर्क केमिकल्स लिमिटेड </td><td> 71491-4 </td><td></td></tr><tr><td> FastBreak </td><td> PROMEGA लिमिटेड </td><td> V8573 </td><td></td></tr><tr><td> Lysonase Bioprocessing अभिकर्मक / 1 मिलीग्राम </td><td> मर्क केमिकल्स लिमिटेड </td><td> 71230 </td><td></td></tr><tr><td> Antifoam 204 </td><td> सिग्मा Aldrich कंपनी लिमिटेड </td><td> A6426 </td><td></td></tr><tr><td> नी - कण MagneHis </td><td> PROMEGA </td><td> V8565 </td><td></td></tr><tr><td> Imidazole </td><td> सिग्मा Aldrich कंपनी लिमिटेड </td><td> 56750 </td><td></td></tr><tr><td> Trizma आधार </td><td> सिग्मा Aldrich Compकिसी भी लिमिटेड </td><td> 93362 </td><td></td></tr><tr><td> NaCl </td><td> VWR </td><td> 27810.295 </td><td></td></tr><tr><td> Bicinchoninic एसिड प्रोटीन दृढ़ संकल्प </td><td> सिग्मा Aldrich कंपनी लिमिटेड </td><td> BCA1 - 1KT </td><td></td></tr><tr><td> दीप खैर प्लेट 2.2 मिलीलीटर स्क्वायर वेल्स पीपी pk10 </td><td> Anachem लिमिटेड </td><td> 1810-00 </td><td></td></tr><tr><td> Microplate 96 microwell अच्छी तरह से फ्लैट नीचे polystyrene का इलाज नहीं 5 प्लेटें स्पष्ट 0.4 ​​मिलीलीटर ही मात्रा 128 मिमी के 12 आस्तीन x 86 मिमी थर्मो वैज्ञानिक Nunc </td><td> फिशर साइंटिफिक लिमिटेड </td><td> जिले-984-090M </td><td></td></tr><tr><td> Microplate ब्लू </td><td> VWR </td><td> NUNC367001 </td><td></td></tr><tr><td> 24 खैर ब्लाकों आरबी (24) </td><td> क्विएज़न </td><td> 19583 </td><td></td></tr><tr><td> Guanidine हाइड्रोक्लोराइड </td><td> VWR </td><td> ALFAA13543.0B </td><td></td></tr><tr><td> TheOnyx के लिए धो सकते हैं सुई </td><td> सन्देशवाहक नौका, समाचार ले जानेवाली नाव GmbH </td><td> 8152-317001 </td><td></td></tr><tr><td> चुंबकीय मोतियों के लिए अभिकर्मक रैक </td><td> सन्देशवाहक नौका, समाचार ले जानेवाली नाव GmbH </td><td> 8152-035003 </td><td></td></tr><tr><td> प्लेट रीडर सिनर्जी HT </td><td> Biotek </td><td> 4200-000043 </td><td></td></tr><tr><td> रोबोट प्लेटफार्म TheOnyx 44OH/150/100 </td><td> सन्देशवाहक नौका, समाचार ले जानेवाली नाव GmbH </td><td> 8145-050046 </td><td></td></tr><tr><td> Microplate शेखर Variomag Teleshaker </td><td> Inheco </td><td> 3800047 </td><td></td></tr><tr><td> 96 अच्छी तरह चुंबक प्रकार एक </td><td> क्विएज़न </td><td> 36915 </td><td></td></tr></tbody></table>

high-throughput purification of affinity-tagged recombinant proteins

X-ray crystallography is the method of choice for obtaining a detailed view of the structure of proteins. Such studies need to be complemented by further biochemical analyses to obtain detailed insights into structure/function relationships. Advances in oligonucleotide- and gene synthesis technology make large-scale mutagenesis strategies increasingly feasible, including the substitution of target residues by all 19 other amino acids. Gain- or loss-of-function phenotypes then allow systematic conclusions to be drawn, such as the contribution of particular residues to catalytic activity, protein stability and/or protein-protein interaction specificity.
In order to attribute the different phenotypes to the nature of the mutation - rather than to fluctuating experimental conditions - it is vital to purify and analyse the proteins in a controlled and reproducible manner. High-throughput strategies and the automation of manual protocols on robotic liquid-handling platforms have created opportunities to perform such complex molecular biological procedures with little human intervention and minimal error rates1-5.
Here, we present a general method for the purification of His-tagged recombinant proteins in a high-throughput manner. In a recent study, we applied this method to a detailed structure-function investigation of TFIIB, a component of the basal transcription machinery. TFIIB is indispensable for promoter-directed transcription in vitro and is essential for the recruitment of RNA polymerase into a preinitiation complex6-8. TFIIB contains a flexible linker domain that penetrates the active site cleft of RNA polymerase9-11. This linker domain confers two biochemically quantifiable activities on TFIIB, namely (i) the stimulation of the catalytic activity during the 'abortive' stage of transcript initiation, and (ii) an additional contribution to the specific recruitment of RNA polymerase into the preinitiation complex4,5,12 . We exploited the high-throughput purification method to generate single, double and triple substitution and deletions mutations within the TFIIB linker and to subsequently analyse them in functional assays for their stimulation effect on the catalytic activity of RNA polymerase4. Altogether, we generated, purified and analysed 381 mutants - a task which would have been time-consuming and laborious to perform manually. We produced and assayed the proteins in multiplicates which allowed us to appreciate any experimental variations and gave us a clear idea of the reproducibility of our results.
This method serves as a generic protocol for the purification of His-tagged proteins and has been successfully used to purify other recombinant proteins. It is currently optimised for the purification of 24 proteins but can be adapted to purify up to 96 proteins.

Watch this Scientific Journal Video about High-throughput Purification of Affinity-tagged Recombinant Proteins at JoVE.com

High-throughput Purification of Affinity-tagged Recombinant Proteins

हम रीकॉम्बीनैंट तरल हैंडलिंग रोबोटिक्स का उपयोग प्रोटीन की आत्मीयता टैग शुद्धि के लिए एक विधि का वर्णन है. इस विधि आम तौर पर एक उच्च throughput प्रारूप में घुलनशील उनकी टैग प्रोटीन के छोटे पैमाने पर शोधन के लिए लागू है.

आत्मीयता टैग पुनः संयोजक प्रोटीन के शोधन उच्च throughput

X-ray crystallography is the method of choice for obtaining a detailed view of the structure of proteins. Such studies need to be complemented by further ...

Research

JoVE Journal

Biology

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पुनः संयोजक प्रोटीन के Methylotrophic खमीर में अभिव्यक्ति Pichia pastoris</em

Protein expression in the microbial eukaryotic host Pichia pastoris offers the possibility to generate high amounts of recombinant protein in a fast and ...

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पुनः संयोजक प्रोटीन का उच्च स्तर क्षणिक अभिव्यक्ति के लिए पौधे के कुशल Agroinfiltration

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के कुशल उत्पादन और शोधन संयोजक Murine Kindlin -3 की biophysical अध्ययन के लिए कीट कोशिकाओं से

Kindlins are essential coactivators, with talin, of the cell surface receptors&#160;integrins and also participate in integrin outside-in signalling, and ...

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में उत्पादित संयोजक Disulfide युक्त जहर प्रोटीन के लिए लागू उच्च Throughput मात्रात्मक अभिव्यक्ति स्क्रीनिंग और शोधन<em&gt; ई. कोली</em

Escherichia coli (E. coli) is the most widely used expression system for the production of recombinant proteins for structural and functional studies. ...

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से पेप्टाइड और प्रोटीन के साथ संयोजक Elastin तरह polypeptides और उनके fusions की गैर chromatographic शोधन<em&gt; कोलाई</em

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