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

这项研究得到了全球前沿研究项目 (NRF-2015M3A6A8065833) 的支持, 并通过韩国政府资助的韩国国家研究基金会 (2018R1A6A1A03024940) (NRF) 进行了基础科学研究项目。

1. 质粒结构
<ol><li>构建一种表达质粒 (pBAD-双 TPL-gfp), 在本构启动子和绿色荧光蛋白 (gfp) 基因的控制下表达柠檬酸杆菌 freundii的 TPL 基因, 并在其控制下对其6标记araBAD 启动子。对于 pBAD-dual-TPL-GFP-E90TAG, 使用站点定向的突变协议, 用琥珀密码子 (标记) 替换多巴的站点 (E90) 的密码子。我们在上一份报告18中描述了建造这种质粒的细节。</li>
	<li>构建 tRNA/aaRS 表达质粒 (pE...

<ol>
	<li>Nagatsu, T., Levitt, M., Udenfriend, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tyrosine+hydroxylase:+The+initial+step+in+norepinephrine+biosynthesis.">Tyrosine hydroxylase: The initial step in norepinephrine biosynthesis.</a> Journal of Biological Chemistry. 239, 2910-2917 (1964).</li><li>Pinder, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Possible+dopamine+derivatives+capable+of+crossing+the+blood-brain+barrier+in+relation+to+parkinsonism.">Possible dopamine derivatives capable of crossing the blood-brain barrier in relation to parkinsonism.</a> Nature. 228 (5269), 358 (1970).</li><li>Waite, J. H., Tanzer, M. 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H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mini-review:+The+role+of+redox+in+DOPA-mediated+marine+adhesion.">Mini-review: The role of redox in DOPA-mediated marine adhesion.</a> Biofouling. 28 (8), 865-877 (2012).</li><li>Silverman, H. G., Roberto, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Understanding+marine+mussel+adhesion.">Understanding marine mussel adhesion.</a> Marine Biotechnology. 9 (6), 661-681 (2007).</li><li>Lee, B. P., Messersmith, P. B., Israelachvili, J. N., Waite, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mussel-inspired+adhesives+and+coatings.">Mussel-inspired adhesives and coatings.</a> Annual Review of Materials Research. 41, 99-132 (2011).</li><li>Umeda, A., Thibodeux, G. N., Zhu, J., Lee, Y., Zhang, Z. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Site-specific+protein+cross-linking+with+genetically+incorporated+3,4-dihydroxy-L-phenylalanine.">Site-specific protein cross-linking with genetically incorporated 3,4-dihydroxy-L-phenylalanine.</a> ChemBioChem. 10 (8), 1302-1304 (2009).</li><li>Umeda, A., Thibodeux, G. N., Moncivais, K., Jiang, F., Zhang, Z. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+versatile+approach+to+transform+low-affinity+peptides+into+protein+probes+with+cotranslationally+expressed+chemical+cross-linker.">A versatile approach to transform low-affinity peptides into protein probes with cotranslationally expressed chemical cross-linker.</a> Analytical Biochemistry. 405 (1), 82-88 (2010).</li><li>Xu, J., Tack, D., Hughes, R. A., Ellington, A. D., Gray, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structure-based+non-canonical+amino+acid+design+to+covalently+crosslink+an+antibody-antigen+complex.">Structure-based non-canonical amino acid design to covalently crosslink an antibody-antigen complex.</a> Journal of Structural Biology. 185 (2), 215-222 (2014).</li><li>Burdine, L., Gillette, T. G., Lin, H. J., Kodadek, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Periodate-triggered+cross-linking+of+DOPA-containing+peptide-protein+complexes.">Periodate-triggered cross-linking of DOPA-containing peptide-protein complexes.</a> Journal of the American Chemical Society. 126 (37), 11442-11443 (2004).</li><li>Borrmann, E., 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=Strain-promoted+oxidation-controlled+cyclooctyne-1,2-quinone+cycloaddition+(SPOCQ)+for+fast+and+activatable+protein+conjugation.">Strain-promoted oxidation-controlled cyclooctyne-1,2-quinone cycloaddition (SPOCQ) for fast and activatable protein conjugation.</a> Bioconjugate Chemistry. 26 (2), 257-261 (2015).</li><li>Ayyadurai, N., 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=Development+of+a+selective,+sensitive,+and+reversible+biosensor+by+the+genetic+incorporation+of+a+metal-binding+site+into+green+fluorescent+protein.">Development of a selective, sensitive, and reversible biosensor by the genetic incorporation of a metal-binding site into green fluorescent protein.</a> Angewandte Chemie International Edition. 50 (29), 6534-6537 (2011).</li><li>Kim, B. J., Cheong, H., Hwang, B. H., Cha, H. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mussel-inspired+protein+nanoparticles+containing+iron(III)-DOPA+complexes+for+pH-responsive+drug+delivery.">Mussel-inspired protein nanoparticles containing iron(III)-DOPA complexes for pH-responsive drug delivery.</a> Angewandte Chemie International Edition. 54 (25), 7318-7322 (2015).</li><li>Alfonta, L., Zhang, Z., Uryu, S., Loo, J. A., Schultz, P. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Site-specific+incorporation+of+a+redox-active+amino+acid+into+proteins.">Site-specific incorporation of a redox-active amino acid into proteins.</a> Journal of the American Chemical Society. 125 (48), 14662-14663 (2003).</li><li>Kim, S., Sung, B. H., Kim, S. C., Lee, H. 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在本议定书中, 介绍了巴胺的生物合成和直接加入。这种方法中使用的细菌细胞可以合成额外的氨基酸, 并将其用作蛋白质合成的非自然构造块。非自然氨基酸的遗传融合一直是生物进化的关键技术, 具有扩展的遗传密码。然而, 这种方法在技术上是不完整的, 正在修改, 以提高整合效率, 并尽量减少扰动的内源翻译系统。最近, 该方法取得了重大进展, 通过重新分配密码子 24,</su...

巴胺是一种氨基酸, 涉及动物和植物中儿茶酚的生物合成。该氨基酸由酪氨酸羟化酶和分子氧 (O2)1合成 Tyr。由于多巴是一大胺的前驱体, 可以渗透血脑屏障, 它已用于治疗帕金森病2。巴比在贻贝黏附蛋白 (地图) 也被发现, 负责贻贝的胶粘剂物产在湿的情况3,4,5,6,7。Tyr 最初是在地图上发现了几巴的位置编码的, 然后被 tyrosinases8、9转换成了巴比。多巴由于其具有有趣的生化特性, 在各种应用中得到了应用。多巴二羟基组化学上易氧化, 氨基酸容易转化为 l-dopaquinone, 黑色素的前驱体。由于其高 electrophilicity, l-dopaquinone 及其衍生物已被用于交联和共轭与硫醇和胺10,11,12,13。12-醌还可以作为 cycloaddition 反应的二烯, 并已用于 bioconjugation 由应变促进氧化控制 cyclooctyne-1,2-quinone (SPOCQ) cycloaddition14。此外, 二羟基集团可以螯合金属离子, 如 Fe3 +和铜2 +, 和含有多巴的蛋白质已用于药物输送和金属离子传感15,16。
多巴通过使用正交氨酰-tRNA (aa-tRNA) 和氨酰-tRNA 合成酶 (aaRS) 对17 , 并用于蛋白质共轭和交联10,11, 被基因纳入蛋白质,12,13。在本报告中, 介绍了从廉价的起始材料中引入 biosynthesized 的实验结果和协议, 以及它在 bioconjugation 中的应用。biosynthesized 使用 TPL, 从邻苯二酚, 丙酮酸和氨的大肠杆菌开始。biosynthesized 多巴是通过表达一个进化的 aa-tRNA 和 aaRS 对巴比直接纳入蛋白质。此外, 含有多巴的 biosynthesized 蛋白与荧光探针结合, 并交联以产生蛋白质寡聚物。这项协议将是有用的蛋白质科学家, biosynthesize 突变蛋白含有一巴和共轭蛋白质与生化探针或药物的工业和制药应用。

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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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			<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 (多巴) 是在动物和植物中儿茶酚胺的生物合成中发现的一种氨基酸。由于氨基酸具有特殊的生化特性, 在生物化学应用中具有多种用途。本报告描述了 biosynthesized 巴的遗传合并协议及其在蛋白质共轭中的应用。多巴是由邻苯二酚、丙酮酸和氨的酪氨酸酚裂解酶 (TPL) biosynthesized 的, 而氨基酸则采用进化后的氨酰-tRNA 和氨酰-tRNA 合成产物对的遗传法直接纳入蛋白质中。这种直接的并网系统有效地吸收了多巴与其他天然氨基酸的很少结合, 并具有更好的蛋白质产量比以前的多巴基因合并系统。多巴多巴蛋白结合具有高效性和特异性, 并显示其对各种应用的实用性。该议定书为蛋白质科学家提供了在所需地点对含有巴胺的突变蛋白进行有效生物合成的详细程序, 以及它们用于工业和制药应用的共轭。

图 1显示了从 TPL 直接并入 biosynthesized 的表达系统。进化的 aa-tRNA 和 aaRS 对的基因被放置在质粒中, 而 gfp 基因 (GFP-E90TAG) 包含琥珀密码子在位置90位于另一个质粒中, 以评估多巴的加入 gfp 荧光。TPL 基因被放置在含有 GFP 基因和组成性表达的同一个表达质粒中, 以最大程度地提高巴比生物合成的产量。利用该表达系统筛选了巴比生物合成的最佳条件。本...

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

Biosynthesized l-dihydroxyphenylalanine (多巴) 的遗传合并及其在蛋白质共轭中的应用

在此, 我们提出了一种从简单的起始材料中 dihydroxyphenylalanine biosynthesized 的遗传方法及其在蛋白质共轭中的应用的协议。

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

A蛋白制备方法在蛋白质与核余子交互使用LC-MS / MS分析的高通量鉴定

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

科研

生物化学

Preparation of Keratin Hydrolysate from Chicken Feathers and Its Application in Cosmetics

鸡羽毛角蛋白水解物的制备及其在化妆品中的应用

Keratin hydrolysates (KHs) are established standard components in hair cosmetics. Understanding the moisturizing effects of KH is advantageous for ...

High Sensitivity Measurement of Transcription Factor-DNA Binding Affinities by Competitive Titration Using Fluorescence Microscopy

荧光显微镜竞争滴定法测定转录机-dna 结合感的高灵敏度

Accurate quantification of transcription factor (TF)-DNA interactions is essential for understanding the regulation of gene expression. Since existing ...

Analysis of Protein Folding, Transport, and Degradation in Living Cells by Radioactive Pulse Chase

放射性脉冲追逐对活细胞蛋白质折叠、迁移和降解的分析

Radioactive pulse-chase labeling is a powerful tool for studying the conformational maturation, the transport to their functional cellular location, and ...

Application of Biolayer Interferometry (BLI) for Studying Protein-Protein Interactions in Transcription

Application of Biolayer Interferometry BLI for Studying Protein-Protein Interactions in Transcription

生物层干涉测量(BLI)在转录中研究蛋白质-蛋白质相互作用的应用

A transcription factor&#160;(TF) is a protein that regulates gene expression by interacting with the RNA polymerase, another TF, and/or template DNA. GrgA ...

PeptiQuick, a One-Step Incorporation of Membrane Proteins into Biotinylated Peptidiscs for Streamlined Protein Binding Assays

PeptiQuick，将膜蛋白一步进生物仿入肽，用于简化蛋白质结合测定

Membrane proteins, including transporters, channels, and receptors, constitute nearly one-fourth of the cellular proteome and over half of current drug ...

Creating Highly Specific Chemically Induced Protein Dimerization Systems by Stepwise Phage Selection of a Combinatorial Single-Domain Antibody Library

通过组合单域抗体库的分步噬菌体选择创建高度特异性化学诱导蛋白分解系统

Protein dimerization events that occur only in the presence of a small-molecule ligand enable the development of small-molecule biosensors for the ...

JUMPn: A Streamlined Application for Protein Co-Expression Clustering and Network Analysis in Proteomics

JUMPn：蛋白质共表达聚类和网络分析在蛋白质组学中的简化应用

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

使用场效应生物传感技术探索分子伴侣Hsp90与其客户蛋白激酶Cdc37之间的生物分子相互作用

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

下拉测定与细菌细胞共表达相结合，作为测试具有挑战性的蛋白质-蛋白质相互作用的省时工具

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