Name: genetic incorporation of biosynthesized l-dihydroxyphenylalanine (dopa) and its application to protein conjugation
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Description: Watch this Scientific Journal Video about Genetic Incorporation of Biosynthesized L-dihydroxyphenylalanine DOPA and Its Application to Protein Conjugation at JoVE.com

इस शोध को ग्लोबल फ्रंटियर रिसर्च प्रोग्राम (एनआरएफ-2015M3A6A8065833), और कोरिया सरकार द्वारा वित्तपोषित नेशनल रिसर्च फाउंडेशन ऑफ कोरिया (एनआरएफ) के माध्यम से बेसिक साइंस रिसर्च प्रोग्राम (2018R1A6A1A03024940) द्वारा समर्थित किया गया ।

1. प्लाज्मिड निर्माण
<ol><li>एक अभिव्यक्ति प्लाज्मिड निर्माण (pBAD-दोहरे TPL-GFP-WT) कि TPL Citrobacter से एक गठन प्रमोटर के नियंत्रण में freundii जीन व्यक्त और ग्रीन फ्लोरोसेंट प्रोटीन (GFP) जीन के नियंत्रण के तहत एक अपने6टैग ...

<ol>
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A protocol for MALDI-MS peptide analysis in proteomics.</a> Analytical Chemistry. 73 (3), 434-438 (2001).</li><li>Mukai, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Highly+reproductive+Escherichia+coli+cells+with+no+specific+assignment+to+the+UAG+codon.">Highly reproductive Escherichia coli cells with no specific assignment to the UAG codon.</a> Scientific Reports. 5, 9699 (2015).</li><li>Lajoie, M. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genomically+recoded+organisms+expand+biological+functions.">Genomically recoded organisms expand biological functions.</a> Science. 342 (6156), 357-360 (2013).</li><li>Bryson, D. 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इस प्रोटोकॉल में, डोपा का संश्लेषण और प्रत्यक्ष शामिल करने का वर्णन किया गया है । इस विधि में प्रयुक्त बैक्टीरियल कोशिका एक अतिरिक्त अमीनो एसिड को संश्लेषित कर सकते हैं और प्रोटीन संश्लेषण के लिए एक अ?...

डोपा एक एमिनो पशुओं और पौधों में catecholamines के संश्लेषण में शामिल एसिड है । इस एमिनो एसिड tyrosine hydroxylase और आणविक ऑक्सीजन (ओ2)1द्वारा Tyr से संश्लेषित है । क्योंकि डोपा डोपामाइन का एक अग्रदूत है और रक्त मस्तिष्क बाधा रिस सकता है, यह पार्किंसंस रोग2के उपचार में इस्तेमाल किया गया है । डोपा भी कौड़ी आसंजन प्रोटीन (नक्शे) है, जो गीला शर्तों3,4,5,6,7में mussels के चिपकने वाले गुणों के लिए जिंमेदार है में पाया जाता है । Tyr शुरू में जहां डोपा नक्शे में पाया जाता है और फिर8,9tyrosinases द्वारा डोपा में बदल दिया है पदों पर इनकोडिंग है । अपने दिलचस्प जैव रासायनिक गुणों के कारण, डोपा आवेदनों की एक किस्म में इस्तेमाल किया गया है । डोपा के dihydroxyl समूह रासायनिक ऑक्सीकरण करने के लिए प्रवण है, और एमिनो एसिड आसानी से एल में परिवर्तित हो जाता है-dopaquinone, मेलेनिन के एक अग्रदूत साबित । अपने उच्च electrophilicity के कारण, एल dopaquinone और उसके डेरिवेटिव thiols और अमीन10,11,12,13के साथ crosslinking और विकार के लिए इस्तेमाल किया गया है । 1, 2-Quinones भी cycloaddition प्रतिक्रियाओं के लिए एक डिएनए के रूप में कार्य कर सकते हैं और bioconjugation के लिए इस्तेमाल किया गया है तनाव से संवर्धित ऑक्सीकरण नियंत्रित cyclooctyne-1, 2-quinone (SPOCQ) cycloaddition14. इसके अलावा, dihydroxyl समूह ऐसे Fe3 + और घन2 +, और डोपा युक्त प्रोटीन के रूप में धातु आयनों chelate कर सकते है दवा वितरण और धातु आयन संवेदन15,16के लिए उपयोग किया गया है ।
डोपा आनुवंशिक रूप से एक ओर्थोगोनल aminoacyl का उपयोग करके प्रोटीन में शामिल किया गया है-tRNA (ए. ए.-tRNA) और aminoacyl-tRNA synthetase (aaRS) जोड़ी17 और प्रोटीन के लिए इस्तेमाल किया विकार और crosslinking10,11, 12,13. इस रिपोर्ट में, प्रयोगात्मक परिणाम और डोपा के आनुवंशिक निगमन के लिए प्रोटोकॉल सस्ते शुरू सामग्री और bioconjugation करने के लिए अपने अनुप्रयोगों से संश्लेषित वर्णित हैं । डोपा एक TPL का उपयोग कर और catechol, पाइरूवेट, और अमोनिया से ई कोलाईमें शुरू करने के लिए संश्लेषित है । संश्लेषित डोपा सीधे एक विकसित ए. ए.-tRNA और डोपा के लिए aaRS जोड़ी व्यक्त द्वारा प्रोटीन में शामिल है । इसके अलावा, डोपा युक्त प्रोटीन है साइट विशेष रूप से एक फ्लोरोसेंट जांच और crosslinked के साथ संयुग्मित के लिए प्रोटीन oligomers का उत्पादन । इस प्रोटोकॉल प्रोटीन वैज्ञानिकों के लिए उपयोगी हो जाएगा, जैव संश्लेषित उत्परिवर्ती प्रोटीन डोपा युक्त है और संयुग्मी रासायनिक जांच या औद्योगिक और दवा अनुप्रयोगों के लिए दवाओं के साथ प्रोटीन ।

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			<td height="21" style="height:21px;width:596px;">1. Plasmid Construction</td>
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			<td height="23" style="height:23px;width:596px;">Plasmid pBAD-dual-TPL-GFP-E90TAG</td>
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			<td>optionally contain the amber stop codon(TAG) at a desired position. Ko, W. et al. Efficient and Site-Specific Antibody Labeling by Strain-promoted Azide-Alkyne Cycloaddition. BKCS. 36 (9), 2352-2354, doi: 10.1002/bkcs.10423, (2015)</td>
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			<td height="21" style="height:21px;width:596px;">Plasmid pEvol-DHPRS2</td>
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			<td style="width:133px;"></td>
			<td>1. Young, T. S., Ahmad, I., Yin, J. A., and Schultz, P. G. An enhanced system for unnatural amino acid mutagenesis in E. coli. J. Mol. Biol. 395 (2), 361-374, doi: 10.1016/j.jmb.2009.10.030, (2010) 2. Kim, S., Sung, B. H., Kim, S. C., Lee, H. S. Genetic incorporation of l-dihydroxyphenylalanine (DOPA) biosynthesized by a tyrosine phenol-lyase. Chem. Commun. 54 (24), 3002-3005, doi: 10.1039/c8cc00281a (2018).</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:596px;">DH10&#946;</td>
			<td style="width:167px;">Invitrogen</td>
			<td style="width:133px;">C6400-03</td>
			<td>Expression Host</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:596px;">Plasmid Mini-prep kit</td>
			<td style="width:167px;">Nucleogen</td>
			<td style="width:133px;">5112</td>
			<td>200/pack</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:596px;">Agarose</td>
			<td style="width:167px;">Intron biotechnology</td>
			<td style="width:133px;">32034</td>
			<td>500 g</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:596px;">Ethidium bromide</td>
			<td style="width:167px;">Alfa Aesar</td>
			<td style="width:133px;">L07482</td>
			<td>1 g</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:596px;">LB Broth</td>
			<td style="width:167px;">BD Difco</td>
			<td style="width:133px;">244620</td>
			<td>500 g</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:596px;">2. Culture preparation</td>
			<td style="width:167px;"></td>
			<td style="width:133px;"></td>
			<td style="width:339px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:596px;">2.1) Electroporation</td>
			<td style="width:167px;"></td>
			<td style="width:133px;"></td>
			<td style="width:339px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:596px;">Micro pulser&#160;</td>
			<td style="width:167px;">BIO-RAD</td>
			<td style="width:133px;">165-2100</td>
			<td style="width:339px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:596px;">Micro pulser cuvette</td>
			<td style="width:167px;">BIO-RAD</td>
			<td style="width:133px;">165-2089</td>
			<td style="width:339px;">0.1 cm electrode gap, pkg. of 50</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:596px;">Ampicillin Sodium</td>
			<td style="width:167px;">Wako</td>
			<td style="width:133px;">018-10372</td>
			<td>25 g</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:596px;">Chloramphenicol</td>
			<td style="width:167px;">Alfa Aesar</td>
			<td style="width:133px;">B20841</td>
			<td>25 g</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:596px;">Agar</td>
			<td style="width:167px;">SAMCHUN</td>
			<td style="width:133px;">214230</td>
			<td>500 g</td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;width:596px;">SOC medium</td>
			<td style="width:167px;">Sigma</td>
			<td style="width:133px;">S1797</td>
			<td>100 mL</td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;width:596px;">3. Expression and Purification of GFP-E90DOPA by biosynthetic system</td>
			<td style="width:167px;"></td>
			<td style="width:133px;"></td>
			<td style="width:339px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;width:596px;">3.1 Expression of GFP-E90DOPA by biosynthetic system</td>
			<td style="width:167px;"></td>
			<td style="width:133px;"></td>
			<td style="width:339px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:596px;">L(+)-Arabinose, 99%</td>
			<td style="width:167px;">Acros</td>
			<td style="width:133px;">104981000</td>
			<td>100 g</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:596px;">Pyrocatechol, 99%</td>
			<td style="width:167px;">SAMCHUN</td>
			<td style="width:133px;">P1387</td>
			<td>25 g</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:596px;">Ammonium sulfate, 99%</td>
			<td style="width:167px;">SAMCHUN</td>
			<td style="width:133px;">A0943</td>
			<td>500 g</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:596px;">pyruvic acid, 98%</td>
			<td style="width:167px;">Alfa Aesar</td>
			<td style="width:133px;">A13875</td>
			<td>100 g</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;width:596px;">Sodium phosphate dibasic, anhydrous, 99%</td>
			<td style="width:167px;">SAMCHUN</td>
			<td style="width:133px;">S0891</td>
			<td>1 kg</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:596px;">Potassium phophate, monobasic, 99%</td>
			<td style="width:167px;">SAMCHUN</td>
			<td style="width:133px;">P1127</td>
			<td>1 kg</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:596px;">Magnesium sulfate, anhydrous, 99%</td>
			<td style="width:167px;">SAMCHUN</td>
			<td style="width:133px;">M0146</td>
			<td>1 kg</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:596px;">D(+)-Glucose, anhydrous, 99%</td>
			<td style="width:167px;">SAMCHUN</td>
			<td style="width:133px;">D0092</td>
			<td>500 g</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:596px;">Glycerol, 99%</td>
			<td style="width:167px;">SAMCHUN</td>
			<td style="width:133px;">G0269</td>
			<td>1 kg</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:596px;">Trace metal mix a5 with co</td>
			<td style="width:167px;">Sigma</td>
			<td style="width:133px;">92949</td>
			<td>25 mL</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:596px;">L-Proline, 99%</td>
			<td style="width:167px;">SAMCHUN</td>
			<td style="width:133px;">P1257</td>
			<td>25 g</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:596px;">L-Phenylalanine, 98.5%</td>
			<td style="width:167px;">SAMCHUN</td>
			<td style="width:133px;">P1982</td>
			<td>25 g</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:596px;">L-Tryptophane</td>
			<td style="width:167px;">JUNSEI</td>
			<td style="width:133px;">49550-0310</td>
			<td>25 g</td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;width:596px;">L-Arginine, 98%</td>
			<td style="width:167px;">SAMCHUN</td>
			<td style="width:133px;">A1149</td>
			<td>25 g</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:596px;">L-Glutamine, 98%</td>
			<td style="width:167px;">JUNSEI</td>
			<td style="width:133px;">27340-0310</td>
			<td>25 g</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:596px;">L-Asparagine monohydrate, 99%</td>
			<td style="width:167px;">SAMCHUN</td>
			<td style="width:133px;">A1198</td>
			<td>25 g</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:596px;">L-Methionine</td>
			<td style="width:167px;">JUNSEI</td>
			<td style="width:133px;">73190-0410</td>
			<td>25 g</td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;width:596px;">L-Histidine hydrochloride monohydrate, 99%</td>
			<td style="width:167px;">SAMCHUN</td>
			<td style="width:133px;">H0604</td>
			<td>25 g</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:596px;">L-Threonine, 99%</td>
			<td style="width:167px;">SAMCHUN</td>
			<td style="width:133px;">T2938</td>
			<td>25 g</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:596px;">L-Leucine</td>
			<td style="width:167px;">JUNSEI</td>
			<td style="width:133px;">87070-0310</td>
			<td>25 g</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:596px;">Glycine, 99%</td>
			<td style="width:167px;">SAMCHUN</td>
			<td style="width:133px;">G0286</td>
			<td>25 g</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:596px;">L-Glutamic acid, 99%</td>
			<td style="width:167px;">SAMCHUN</td>
			<td style="width:133px;">G0233</td>
			<td>25 g</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:596px;">L-Alanine, 99%</td>
			<td style="width:167px;">SAMCHUN</td>
			<td style="width:133px;">A1543</td>
			<td>25 g</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:596px;">L-Isoleucine, 99%</td>
			<td style="width:167px;">SAMCHUN</td>
			<td style="width:133px;">I1049</td>
			<td>25 g</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:596px;">L-Valine, 99%</td>
			<td style="width:167px;">SAMCHUN</td>
			<td style="width:133px;">V0088</td>
			<td>25 g</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:596px;">L-Serine</td>
			<td style="width:167px;">SAMCHUN</td>
			<td style="width:133px;">S2447</td>
			<td>25 g</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:596px;">L-Aspartic acid</td>
			<td style="width:167px;">SAMCHUN</td>
			<td style="width:133px;">A1205</td>
			<td>25 g</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:596px;">L-Lysine monohydrochloride, 99%</td>
			<td style="width:167px;">SAMCHUN</td>
			<td style="width:133px;">L0592</td>
			<td>25 g</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:596px;">3.2 Cell lysis</td>
			<td style="width:167px;"></td>
			<td style="width:133px;"></td>
			<td style="width:339px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:596px;">Imidazole, 99%</td>
			<td style="width:167px;">SAMCHUN</td>
			<td style="width:133px;">I0578</td>
			<td>1kg</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:596px;">Sodium phosphate monobasic, 98%</td>
			<td style="width:167px;">SAMCHUN</td>
			<td style="width:133px;">S0919</td>
			<td>1 kg</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:596px;">Sodium Chloride, 99%</td>
			<td style="width:167px;">SAMCHUN</td>
			<td style="width:133px;">S2907</td>
			<td>1 kg</td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;width:596px;">Ultrasonic Processor - 150 microliters to 150 milliliters</td>
			<td style="width:167px;">SONIC &#38; MATERIALS</td>
			<td style="width:133px;">VCX130</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:596px;">3.3 Ni-NTA Affinity Chromatography</td>
			<td style="width:167px;"></td>
			<td style="width:133px;"></td>
			<td style="width:339px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:596px;">Ni-NTA resin</td>
			<td style="width:167px;">QIAGEN</td>
			<td style="width:133px;">30210</td>
			<td>25 mL</td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;width:596px;">Polypropylene column</td>
			<td style="width:167px;">QIAGEN</td>
			<td style="width:133px;">34924</td>
			<td>50/pack, 1 mL capacity</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;width:596px;">4. Oligomerization of Purified GFP-E90DOPA&#160;</td>
			<td style="width:167px;"></td>
			<td style="width:133px;"></td>
			<td style="width:339px;"></td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;width:596px;">Sodium periodate, 99.8&#38;</td>
			<td style="width:167px;">Acros</td>
			<td style="width:133px;">419610050</td>
			<td>5 g</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;width:596px;">5. Conjugation of GFP-E90DOPA with an Alkyne Probe by Strain-Promoted Oxidation-Controlled Cyclooctyne&#8211;1,2-Quinone Cycloaddition (SPOCQ)&#160;</td>
			<td style="width:167px;"></td>
			<td style="width:133px;"></td>
			<td style="width:339px;"></td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;width:596px;">Cy5.5-ADIBO&#160;</td>
			<td style="width:167px;">FutureChem</td>
			<td style="width:133px;">FC-6119</td>
			<td>1mg</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:596px;">6. Purification of Labeled GFP</td>
			<td style="width:167px;"></td>
			<td style="width:133px;"></td>
			<td style="width:339px;"></td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;width:596px;">Amicon Ultra 0.5 mL Centrifugal Filters</td>
			<td style="width:167px;">MILLIPORE</td>
			<td style="width:133px;">UFC500396</td>
			<td>96/pack, 500ul capacity</td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;width:596px;">7. SDS-PAGE Analysis and Fluorescence Gel Scanning</td>
			<td style="width:167px;"></td>
			<td style="width:133px;"></td>
			<td style="width:339px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:596px;">1,4-Dithio-DL-threitol, DTT, 99.5 %</td>
			<td style="width:167px;">Sigma</td>
			<td style="width:133px;">10708984001</td>
			<td>10 g</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:596px;">NuPAGE LDS Sample Buffer, 4X</td>
			<td style="width:167px;">Thermofisher</td>
			<td style="width:133px;">NP0007</td>
			<td>10 mL</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:596px;">MES running buffer</td>
			<td style="width:167px;">Thermofisher</td>
			<td style="width:133px;">NP0002</td>
			<td>500 mL</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:596px;">Nupage Novex 4-12% SDS PAGE gels</td>
			<td style="width:167px;">Thermofisher</td>
			<td style="width:133px;">NO0321</td>
			<td>12 well</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:596px;">Coomassie Brilliant Blue R-250</td>
			<td style="width:167px;">Wako</td>
			<td style="width:133px;">031-17922</td>
			<td>25 g</td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;width:596px;">G:BOX Chemi Fluorescent &#38; Chemiluminescent Imaging System</td>
			<td style="width:167px;">Syngene</td>
			<td style="width:133px;"></td>
			<td>G BOX Chemi XT4</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;width:596px;">8. MALDI-TOF MS analysis by Trypsin Digestion</td>
			<td style="width:167px;"></td>
			<td style="width:133px;"></td>
			<td style="width:339px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;width:596px;">8.1 Preparation of the digested peptide sample by trypsin digestion</td>
			<td style="width:167px;"></td>
			<td style="width:133px;"></td>
			<td style="width:339px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:596px;">Tris(hydroxymethyl)aminomethane, 99%</td>
			<td style="width:167px;">SAMCHUN</td>
			<td style="width:133px;">T1351</td>
			<td>500 g</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:596px;">Hydrochloric acid, 35~37%</td>
			<td style="width:167px;">SAMCHUN</td>
			<td style="width:133px;">H0256</td>
			<td>500 mL</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:596px;">Dodecyl sulfate sodium salt, 85%</td>
			<td style="width:167px;">SAMCHUN</td>
			<td style="width:133px;">D1070</td>
			<td>250 g</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:596px;">Iodoacetamide</td>
			<td style="width:167px;">Sigma</td>
			<td style="width:133px;">I6125</td>
			<td>5 g</td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;width:596px;">Trypsin Protease, MS Grade</td>
			<td style="width:167px;">Thermofisher</td>
			<td style="width:133px;">90057</td>
			<td>5 x 20 &#181;g/pack</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:596px;">C-18 spin columns</td>
			<td style="width:167px;">Thermofisher</td>
			<td style="width:133px;">89870</td>
			<td>25/pack, 200 &#181;L capacity</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;width:596px;">8.2 Analysis of the digested peptide by MALDI-TOF</td>
			<td style="width:167px;"></td>
			<td style="width:133px;"></td>
			<td style="width:339px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:596px;">Acetonitirile, 99.5%</td>
			<td style="width:167px;">SAMCHUN</td>
			<td style="width:133px;">A0125</td>
			<td>500 mL</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:596px;">&#945;-Cyano-4-hydroxycinnamic acid</td>
			<td style="width:167px;">Sigma</td>
			<td style="width:133px;">C2020</td>
			<td>10 g</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:596px;">Trifluoroacetic acid, 99%</td>
			<td style="width:167px;">SAMCHUN</td>
			<td style="width:133px;">T1666</td>
			<td>100 g</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:596px;">MTP 384 target plate ground steel BC targets</td>
			<td style="width:167px;">Bruker</td>
			<td style="width:133px;">8280784</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:596px;">Bruker Autoflex Speed MALDI-TOF mass spectrometer</td>
			<td style="width:167px;">Bruker</td>
			<td style="width:133px;"></td>
			<td></td>
		</tr>
	</tbody>
</table>

genetic incorporation of biosynthesized l-dihydroxyphenylalanine (dopa) and its application to protein conjugation

L-dihydroxyphenylalanine (डोपा) एक एमिनो एसिड पशुओं और पौधों में catecholamines के संश्लेषण में पाया जाता है । अपने विशेष जैव रासायनिक गुणों की वजह से, एमिनो एसिड जैव रासायनिक अनुप्रयोगों में कई का उपयोग करता है । इस रिपोर्ट में प्रोटीन विकार के लिए एक संश्लेषित डोपा और उसके आवेदन के जेनेटिक शामिल करने के लिए एक प्रोटोकॉल का वर्णन है । डोपा एक tyrosine phenol द्वारा संश्लेषित है-lyase (TPL) से catechol, पाइरूवेट, और अमोनिया, और एमिनो एसिड सीधे प्रोटीन में आनुवंशिक निगमन विधि द्वारा एक विकसित aminoacyl-tRNA और aminoacyl-tRNA synthetase जोड़ी का उपयोग कर शामिल है । यह प्रत्यक्ष निगमन प्रणाली कुशलतापूर्वक अंय प्राकृतिक अमीनो एसिड के थोड़ा निगमन और डोपा के लिए पिछले आनुवंशिक निगमन प्रणाली की तुलना में बेहतर प्रोटीन उपज के साथ डोपा शामिल हैं । प्रोटीन विकार डोपा युक्त प्रोटीन के साथ कुशल और साइट विशिष्ट है और विभिंन अनुप्रयोगों के लिए अपनी उपयोगिता से पता चलता है । इस प्रोटोकॉल के लिए वांछित साइटों पर डोपा युक्त उत्परिवर्ती प्रोटीन के कुशल और औद्योगिक और दवा अनुप्रयोगों के लिए उनके विकार में शामिल करने के लिए विस्तृत प्रक्रियाओं के साथ प्रोटीन वैज्ञानिकों प्रदान करता है ।

एक TPL से संश्लेषित डोपा के प्रत्यक्ष शामिल करने के लिए अभिव्यक्ति प्रणाली चित्रा 1में दिखाया गया है । विकसित ए. ए. के लिए जीन-tRNA और aaRS जोड़ी एक प्लाज्मिड में रखा जाता है, और GFP जीन (GFP-E90TAG) ?...

Watch this Scientific Journal Video about Genetic Incorporation of Biosynthesized L-dihydroxyphenylalanine DOPA and Its Application to Protein Conjugation at JoVE.com

Genetic Incorporation of Biosynthesized L-dihydroxyphenylalanine DOPA and Its Application to Protein Conjugation

आनुवंशिक संश्लेषित एल-dihydroxyphenylalanine (डोपा) और प्रोटीन के लिए अपने आवेदन विकार

यहां, हम एल के आनुवंशिक शामिल करने के लिए एक प्रोटोकॉल वर्तमान dihydroxyphenylalanine सरल प्रारंभिक सामग्री और प्रोटीन विकार के लिए अपने आवेदन से संश्लेषित ।

L-dihydroxyphenylalanine (DOPA) is an amino acid found in the biosynthesis of catecholamines in animals and plants. Because of its particular biochemical ...

Research

JoVE Journal

Biochemistry

Genetic Incorporation of Biosynthesized L-dihydroxyphenylalanine (DOPA) and Its Application to Protein Conjugation

A Protein Preparation Method for the High-throughput Identification of Proteins Interacting with a Nuclear Cofactor Using LC-MS/MS Analysis

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