Name: genetic incorporation of biosynthesized l-dihydroxyphenylalanine (dopa) and its application to protein conjugation
Uploaded: 2018-08-24T15:00:00
Description: Watch this Scientific Journal Video about Genetic Incorporation of Biosynthesized L-dihydroxyphenylalanine DOPA and Its Application to Protein Conjugation at JoVE.com

Questa ricerca è stata sostenuta dal programma di ricerca di frontiera internazionale (NRF-2015M3A6A8065833), e programma di ricerca scienza base (2018R1A6A1A03024940) attraverso National Research Foundation di Corea (NRF) finanziato dal governo della Corea.

1. plasmide costruzione
<ol><li>Costruire un plasmide di espressione (pBAD-dual-TPL-GFP-WT) che esprime il gene TPL dal freundii del citrobatterio sotto il controllo di un promotore costitutivo e il gene della proteina fluorescente verde (GFP) con un suo6-tag sotto il controllo della promotore araBAD. Per pBAD-dual-TPL-GFP-E90TAG, sostituire il codone per il sito (E90) di DOPA con un codone ambrato (TAG), utilizzando un protocollo di mutagenesi sito-diretta. I dettagli per la costruzione di questo p...

<ol>
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In questo protocollo, la biosintesi e l'incorporazione diretta della DOPA sono descritti. La cellula batterica utilizzata in questo metodo può sintetizzare un aminoacido ulteriore e usarlo come un innaturale building block per la sintesi proteica. La costituzione genetica di amminoacidi non naturali è stata una tecnologia chiave per lo sviluppo dell'organismo innaturale con un codice genetico espanso. Tuttavia, questo metodo è stato tecnicamente incompleto e viene modificato per migliorare l'efficienza di incorporazio...

DOPA è un aminoacido coinvolto nella biosintesi delle catecolamine in animali e piante. Questo aminoacido è sintetizzato da Tyr idrossilasi della tirosina e ossigeno molecolare (O2)1. Perché DOPA è un precursore della dopamina e può permeare la barriera emato - encefalica, è stato utilizzato nel trattamento della malattia di Parkinson2. DOPA si trova anche in proteine di adesione di cozza (mappe), che sono responsabili per le proprietà adesive di cozze in condizioni di bagnato3,4,5,6,7. Tyr è inizialmente codificato nelle posizioni dove DOPA si trova nelle mappe e viene quindi convertito in DOPA da tirosinasi8,9. Grazie alle sue interessanti proprietà biochimiche, DOPA è stato utilizzato in una varietà di applicazioni. Il gruppo di dihydroxyl della DOPA è chimicamente incline all'ossidazione e l'aminoacido è facilmente convertito in L-dopachinone, un precursore di melanine. A causa della sua elevata electrophilicity, L-dopaquinone e suoi derivati sono stati utilizzati per la reticolazione e la coniugazione con tioli e ammine10,11,12,13. 1,2-chinoni possono anche funzionare come un diene per reazioni di cicloaddizione e sono stati utilizzati per bioconjugation dal ceppo-promosso ossidazione controllata cyclooctyne-1,2-chinone (SPOCQ) cicloaddizione14. Inoltre, il gruppo di dihydroxyl possa chelare ioni metallici come Fe3 + e Cu2 +, e proteine contenenti DOPA sono stati utilizzati per la consegna della droga e dello ione del metallo rilevamento15,16.
DOPA è stato incorporato nelle proteine utilizzando un ortogonale aminoacyl-tRNA (aa-tRNA) e amminoacil-tRNA sintetasi (aaRS) coppia17 geneticamente e utilizzato per proteina coniugazione e reticolazione10,11, 12,13. In questo rapporto, i risultati sperimentali e protocolli per la costituzione genetica di DOPA biosynthesized da materie prime a buon mercato e le sue applicazioni per bioconjugation sono descritti. DOPA biosynthesized utilizzando un TPL e a partire da catecolo, piruvato e ammoniaca in Escherichia coli. Biosynthesized DOPA è direttamente incorporato nelle proteine esprimendo una coppia di aa-tRNA e aaRS evoluta per DOPA. Inoltre, la proteina biosynthesized contenenti DOPA site-specifically è coniugata con una sonda fluorescente e reticolati per produrre oligomeri di proteina. Questo protocollo sarà utile per gli scienziati di proteina, di provitamina proteine mutanti contenenti DOPA e coniugato le proteine con sonde biochimici o farmaci per applicazioni industriali e farmaceutiche.

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			<td height="21" style="height:21px;width:596px;">1. Plasmid Construction</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;">Plasmid pBAD-dual-TPL-GFP-E90TAG</td>
			<td style="width:167px;"></td>
			<td style="width:133px;"></td>
			<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>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:596px;">Plasmid pEvol-DHPRS2</td>
			<td style="width:167px;"></td>
			<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>
		</tr>
		<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>
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			<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>
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		<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>
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			<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-diidrossifenilalanina (DOPA) è un aminoacido presente nella biosintesi delle catecolamine in animali e piante. Grazie alle sue particolari proprietà biochimiche, l'amminoacido ha molteplici usi in applicazioni biochimiche. Questo rapporto descrive un protocollo per la costituzione genetica di biosynthesized DOPA e sua applicazione alla coniugazione di proteina. DOPA biosynthesized da una tirosina fenolo-liasi (TPL) da catecolo, piruvato e ammoniaca, e l'aminoacido è direttamente incorporato nelle proteine con il metodo di incorporazione genetico utilizza un'evoluta amminoacil-tRNA e coppia di aminoacyl-tRNA sintetasi. Questo sistema di incorporazione diretta in modo efficiente incorpora DOPA con piccolo incorporazione di altri amminoacidi naturali e con migliore rendimento di proteine rispetto al precedente sistema di costituzione genetica per DOPA. Coniugazione di proteine con proteine contenenti DOPA è efficiente e site-specific e dimostra la sua utilità per varie applicazioni. Questo protocollo fornisce agli scienziati di proteina con procedure dettagliate per la biosintesi efficiente delle proteine mutanti contenenti DOPA nei siti desiderati e loro coniugazione per applicazioni industriali e farmaceutiche.

Il sistema di espressione per l'incorporazione diretta di DOPA biosynthesized da un TPL è illustrato nella Figura 1. I geni per la coppia di aa-tRNA e aaRS evoluto sono collocati in un plasmide, e il gene GFP (GFP-E90TAG) contenenti un codone ambrato alla posizione 90 si trova in un altro plasmide per valutare l'incorporazione di DOPA di fluorescenza di GFP. Il gene TPL è collocato nel plasmide stessa espressione che contiene il gene GFP e costitutivamente ...

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

Genetiche incorporazione di Biosynthesized L-diidrossifenilalanina (DOPA) e la sua applicazione alla proteina coniugazione

Qui, presentiamo un protocollo per la costituzione genetica di L-diidrossifenilalanina biosynthesized da semplici materiali di partenza e la sua applicazione alla coniugazione di proteina.

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

Una proteina metodo di preparazione per l&#39;high-throughput identificazione di proteine ​​che interagiscono con un cofattore nucleare Utilizzando LC-MS / MS Analysis

Transcriptional coregulators are vital to the efficient transcriptional regulation of nuclear chromatin structure. Coregulators play a variety of roles in ...

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With recent advances in mass spectrometry-based proteomics technologies, deep profiling of hundreds of proteomes has become increasingly feasible. ...

Exploring Biomolecular Interaction Between the Molecular Chaperone Hsp90 and Its Client Protein Kinase Cdc37 using Field-Effect Biosensing Technology

Esplorare l'interazione biomolecolare tra lo chaperone molecolare Hsp90 e la sua proteina chinasi cliente Cdc37 utilizzando la tecnologia di biorilevamento ad effetto di campo

Biomolecular interactions play versatile roles in numerous cellular processes&#160;by regulating and coordinating functionally relevant biological events. ...

Pulldown Assay Coupled with Co-Expression in Bacteria Cells as a Time-Efficient Tool for Testing Challenging Protein-Protein Interactions

Saggio di pulldown accoppiato con co-espressione in cellule batteriche come strumento efficiente in termini di tempo per testare interazioni proteina-proteina impegnative

Pulldown is an easy and widely used protein-protein interaction assay. However, it has limitations in studying protein complexes that do not assemble ...