Name: genetic incorporation of biosynthesized l-dihydroxyphenylalanine (dopa) and its application to protein conjugation
Uploaded: 2018-08-24T15:00:00.000+00:00
Duration: 10 min 24 s
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 (pEVOL-DHPRS2)18,19用于多巴基因的合并。pEVOL 是一种特殊的质粒载体, 表达了两个 aaRS 基因的独立启动子, 并在前一份报告中描述了质粒的详细信息19。</li>
	<li>使用商业上可用的质粒制备试剂盒获得高纯度质粒 dna。如有必要, 用琼脂糖凝胶电泳检查准备的 dna 纯度。</li>
</ol>2. 文化准备
<ol><li>
穿孔
	<ol>
<li>用电控大肠杆菌DH10β菌株进行实验。将每种质粒 DNA 的1µL (pEVOL-DHPRS2 和 pBAD-dual-TPL-GFP-E90TAG) 添加到20µL 的合格细胞中。用吸管将它们轻轻地混合成均匀的混合物。</li>
		<li>将混合物转移到电穿孔小试管。把试管放在电穿孔室里。将试管推入室内, 使试管室接触牢固。</li>
		<li>冲击试管, 使用25µF 和2.5 伏的0.1 厘米小试管。</li>
		<li>加入1毫升的 prewarmed 超级最佳汤 (SOC), 含有 2% (w/v) 胰蛋白胨, 0.5% (瓦特/v) 酵母提取物, 10 毫米氯化钠, 2.5 毫米氯化钾, 10 毫米氯化镁2, 和20毫米葡萄糖, 到试管, 并转移细胞到培养管。用颤抖的方法在37摄氏度的1小时内孵化, 以拯救细胞。</li>
		<li>在含有35µg/毫升氯霉素和100µg/毫升氨苄西林的溶源性汤 (LB) 琼脂板上传播 10-100 µL 的获救细胞。孵育细胞在37°c 16 小时。</li>
	</ol>
</li>
	<li>
起动机培养制剂
	<ol>
<li>接种5毫升含有抗生素的 LB 培养基中的单个转化的菌落。用颤抖的方法在37摄氏度孵化出12小时的蚁群。</li>
	</ol>
</li>
</ol>3. 生物合成系统对 GFP-E90DOPA 的表达和纯化
<ol><li>
生物合成系统 GFP-E90DOPA 的表达
	<ol>
<li>将起始培养 (2 毫升) 转移到100毫升的 PA-5052 中等20 (50 毫米 Na2HPO4, 50 毫米2PO4, 25 毫米 [NH4]2所以4, 2 毫米 MgSO4, 0.1% [w/v] 痕量金属, 0.5% [瓦特/v] 甘油,0.05% [瓦特/v] 葡萄糖, 和 5% [w/v] 氨基酸 [pH 7.25]) 含有35µg/毫升氯霉素, 100 µg/毫升氨苄西林, 100 毫米丙酮酸, 10 毫米邻苯二酚, 25 毫米氨, 300 µM dithiothreitol (德勤) 其次是孵化在30°c 与震动。</li>
		<li>当光学密度 (OD) 在550毫微米达到0.8 时, 加入2毫升 0.2% (瓦特/v) l-阿拉伯糖。用颤抖的方法在30摄氏度孵化样品12小时。</li>
		<li>旋转 1.1万 x g 的细胞5分钟, 丢弃上清, 并冻结细胞颗粒在-80 摄氏度。</li>
	</ol>
</li>
	<li>
细胞裂解
	<ol>
<li>在冰上解冻细胞颗粒, 并用重悬10毫升的溶解缓冲液中含有50毫米 NaH2PO4 (pH 8.0), 10 毫米咪唑和300毫米氯化钠的细胞。</li>
		<li>油脂实验悬浮细胞在冰上的10分钟使用80% 超声波振幅与脉冲二十五年代和三十五年代。</li>
		<li>在 1.8万 x g 4 摄氏度处旋转细胞裂解液15分钟. 将上清液转移到新鲜的试管中进行提纯并丢弃颗粒。</li>
	</ol>
</li>
	<li>
镍-NTA 亲和层析法
	<ol>
<li>加入镍 NTA (nitrilotriacetic 酸) 树脂 (300 µL 树脂悬浮液100毫升培养) 到上清, 并将蛋白质结合到4摄氏度的镍 NTA 树脂上, 轻轻摇动悬浮液 1 h。</li>
		<li>将悬浮液转移到聚丙烯柱上, 用5毫升的洗涤缓冲液洗涤树脂 3x, 其中含有50毫米 NaH2PO4 (pH 8.0)、20毫米咪唑和300毫米氯化钠。</li>
		<li>洗脱蛋白质3x 与300µL 洗脱缓冲器包含50毫米 NaH2PO4 (pH 8.0), 250 毫米咪唑和300毫米氯化钠。</li>
		<li>通过测量 280 nm 的吸光度测定蛋白质浓度。用蛋白质消光系数计算器 (如https://web.expasy.org/protparam/) 和独立测量的 GFP-E90DOPA 计算了 280 nm 的消光系数 (24630 米-1厘米-1);coefficiennt (2630 米-1厘米-1) 为巴胺。作为一种替代方法, 使用布拉德福德蛋白21测定蛋白质浓度。</li>
	</ol>
</li>
</ol>4. 纯化 GFP-E90DOPA 的齐聚
<ol><li>在含20毫米 Na300GFP-E90DOPA50 (pH 2) 和4毫米氯化钠的磷酸盐缓冲液中, 添加1µL 的高碘酸钠在 H2O (6 毫米) (1.0 equiv) 到 GFP-E90DOPA) 到8.0 µM (300 HPO) 的溶液中。</li>
	<li>允许齐聚反应以25摄氏度为48小时进行。</li>
	<li>用月桂酸钠-聚丙烯酰胺凝胶电泳法分析反应混合物 (SDS 页), 如步骤6所述。</li>
</ol>5. GFP-E90DOPA 与炔烃探针的共轭 SPOCQ
<ol><li>在 H2O (4.0 毫米) (10 ADIBO) 中添加1µL Cy5.5 链接的 azadibenzocyclooctyne (Cy5.5 equiv)22 . GFP-E90DOPA) 到溶液中的20µL 的 GFP-E90DOPA (20 µM) 在磷酸盐缓冲器含有50毫米 Na2HPO4 (pH 8.0) 和300毫米氯化钠。</li>
	<li>将高碘酸钠的1µL 在 H2O (400 µM) (1.0 equiv GFP-E90DOPA) 中加入反应混合物。</li>
	<li>允许 SPOCQ 反应在25摄氏度进行1小时。如果使用光敏感探头, 请用铝箔覆盖反应容器。</li>
</ol>6. 标记的 GFP 的纯化
<ol><li>通过添加磷酸盐缓冲剂稀释 SPOCQ 反应混合物, 含有50毫米 Na2HPO4 (pH 8.0) 和300毫米氯化钠, 最多500µL。</li>
	<li>将稀释后的溶液转移到离心过滤器旋压柱上。离心机的旋转柱在 1.4万 x g在4°c 15 分钟, 并放弃流经。重复此步骤 3x, 以删除任何多余的 Cy5.5-ADIBO。</li>
	<li>将纯化样品从自旋柱转移到1.5 毫升离心管。</li>
	<li>另外, 对同一缓冲液进行透析纯化。</li>
	<li>将纯化的 GFP 保存在4摄氏度。</li>
</ol>7. SDS 页分析和荧光凝胶扫描
<ol><li>通过添加5µL 蛋白质样品缓冲器 (见材料表) 和2µL (1.00 米) 到13µL 的纯化, 或粗, 标记为 GFP (13.6 µM 或0.38 毫克/毫升), 为 SDS 页分析准备蛋白质样品。对于寡聚 gfp 的分析, 使用更高浓度的 gfp (67.8 µM 或1.9 毫克/毫升)。变性蛋白质的孵化, 在95°c 10 分钟。</li>
	<li>将 4%-12% 双三页凝胶盒 (购买的预制凝胶盒) 放置到电泳单元中, 并添加一个正在运行的缓冲器 (见材料表)。加载蛋白质样品和分子量标记。在 200 v 进行电泳 35-40 分钟。 注意: 通过在黑暗中执行所有的过程, 避免对蛋白质样品的任何曝光, 以尽量减少荧光探针的光漂白。</li>
	<li>使用荧光扫描仪进行荧光成像。将蛋白质凝胶从电泳中包裹起来, 放在荧光扫描仪上。使用适当的波长设置扫描凝胶。对于 Cy5.5, 请选择扫描仪软件中提供的 Cy5 模式。</li>
	<li>用商业蛋白染色试剂染色凝胶。</li>
</ol>8. 胰蛋白酶消化 MALDI 分析
<ol><li>孵育100µL 的纯化 GFP-E90DOPA (2.2 毫克/毫升或80µM) 在一个缓冲区中含有50毫米三 HCl (pH 8.0), 0.1% (w/v) SDS, 25 毫米, 在60°c 为 1 h。</li>
	<li>在2O (IAA, 4.0 米) (500 equiv GFP-E90DOPA) 中添加1µL Iodoacetamide 在同一缓冲区中的纯化 GFP-E90DOPA 溶液 (80 µM)。在25摄氏度的混合物中孵育30分钟, 保护光线。</li>
	<li>通过加入胰蛋白酶 (1:100 蛋白酶到基质蛋白比值 [w/w]) 来消化 GFP-E90DOPA 样品, 并在37摄氏度的4小时内孵化反应混合物。</li>
	<li>采用 C18自旋柱的标准脱盐法纯化消化肽样品。</li>
	<li>以 alpha-cyano-4-hydroxycinnamic 酸 (CHCA) 为基质23, 通过对基质辅助激光解吸/电离飞行时间质谱 (MALDI) 的报告协议, 对消化肽进行分析。</li>
</ol>

<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. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polyphenolic+substance+of+mytilus+edulis:+novel+adhesive+containing+L-DOPA+and+hydroxyproline.">Polyphenolic substance of mytilus edulis: novel adhesive containing L-DOPA and hydroxyproline.</a> Science. 212 (4498), 1038-1040 (1981).</li><li>Lee, H., Scherer, N. F., Messersmith, P. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-molecule+mechanics+of+mussel+adhesion.">Single-molecule mechanics of mussel adhesion.</a> Proceedings of the National Academy of Sciences of the United States of America. 103 (35), 12999-13003 (2006).</li><li>Papov, V. V., Diamond, T. V., Biemann, K., 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=Hydroxyarginine-containing+polyphenolic+proteins+in+the+adhesive+plaques+of+the+marine+mussel+Mytilus+edulis.">Hydroxyarginine-containing polyphenolic proteins in the adhesive plaques of the marine mussel Mytilus edulis.</a> Journal of Biological Chemistry. 270 (34), 20183-20192 (1995).</li><li>Waite, J. H., Qin, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polyphosphoprotein+from+the+adhesive+pads+of+Mytilus+edulis.">Polyphosphoprotein from the adhesive pads of Mytilus edulis.</a> Biochemistry. 40 (9), 2887-2893 (2001).</li><li>Nicklisch, S. C., 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=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. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+incorporation+of+L-dihydroxyphenylalanine+(DOPA)+biosynthesized+by+a+tyrosine+phenol-lyase.">Genetic incorporation of L-dihydroxyphenylalanine (DOPA) biosynthesized by a tyrosine phenol-lyase.</a> Chemical Communications. 54 (24), 3002-3005 (2018).</li><li>Young, T. S., Ahmad, I., Yin, 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=An+enhanced+system+for+unnatural+amino+acid+mutagenesis+in+E.+coli.">An enhanced system for unnatural amino acid mutagenesis in E. coli.</a> Journal of Molecular Biology. 395 (2), 361-374 (2010).</li><li>Studier, F. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+production+by+auto-induction+in+high+density+shaking+cultures.">Protein production by auto-induction in high density shaking cultures.</a> Protein Expression and Purification. 41 (1), 207-234 (2005).</li><li>Bradford, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+rapid+and+sensitive+method+for+the+quantitation+of+microgram+quantities+of+protein+utilizing+the+principle+of+protein-dye+binding.">A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding.</a> Analytical Biochemistry. 72 (1-2), 248-254 (1976).</li><li>Jang, S., Sachin, K., Lee, H. J., Kim, D. W., Lee, H. S. <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+simple+method+for+protein+conjugation+by+copper-free+click+reaction+and+its+application+to+antibody-free+Western+blot+analysis.">Development of a simple method for protein conjugation by copper-free click reaction and its application to antibody-free Western blot analysis.</a> Bioconjugate Chemistry. 23 (11), 2256-2261 (2012).</li><li>Gobom, 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=Alpha-cyano-4-hydroxycinnamic+acid+affinity+sample+preparation.+A+protocol+for+MALDI-MS+peptide+analysis+in+proteomics.">Alpha-cyano-4-hydroxycinnamic acid affinity sample preparation. 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. I., 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=Continuous+directed+evolution+of+aminoacyl-tRNA+synthetases.">Continuous directed evolution of aminoacyl-tRNA synthetases.</a> Nature Chemical Biology. 13 (12), 1253-1260 (2017).</li><li>Guo, J., Melancon, C. E., Lee, H. S., Groff, D., 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=Evolution+of+amber+suppressor+tRNAs+for+efficient+bacterial+production+of+proteins+containing+nonnatural+amino+acids.">Evolution of amber suppressor tRNAs for efficient bacterial production of proteins containing nonnatural amino acids.</a> Angewandte Chemie International Edition. 48 (48), 9148-9151 (2009).</li><li>Lee, S., 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=A+facile+strategy+for+selective+phosphoserine+incorporation+in+histones.">A facile strategy for selective phosphoserine incorporation in histones.</a> Angewandte Chemie International Edition. 52 (22), 5771-5775 (2013).</li><li>Neumann, H., Wang, K., Davis, L., Garcia-Alai, M., Chin, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Encoding+multiple+unnatural+amino+acids+via+evolution+of+a+quadruplet-decoding+ribosome.">Encoding multiple unnatural amino acids via evolution of a quadruplet-decoding ribosome.</a> Nature. 464 (7287), 441-444 (2010).</li><li>Lang, K., Chin, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bioorthogonal+reactions+for+labeling+proteins.">Bioorthogonal reactions for labeling proteins.</a> ACS Chemical Biology. 9 (1), 16-20 (2014).</li><li>Lang, K., Chin, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+Incorporation+of+unnatural+amino+acids+and+bioorthogonal+labeling+of+proteins.">Cellular Incorporation of unnatural amino acids and bioorthogonal labeling of proteins.</a> Chemical Reviews. 114 (9), 4764-4806 (2014).</li><li>Plass, T., Milles, S., Koehler, C., Schultz, C., Lemke, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetically+encoded+copper-free+click+chemistry.">Genetically encoded copper-free click chemistry.</a> Angewandte Chemie International Edition. 50 (17), 3878-3881 (2011).</li><li>Seitchik, J. L., 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=Genetically+encoded+tetrazine+amino+acid+directs+rapid+site-specific+in+vivo+bioorthogonal+ligation+with+trans-cyclooctenes.">Genetically encoded tetrazine amino acid directs rapid site-specific in vivo bioorthogonal ligation with trans-cyclooctenes.</a> Journal of the American Chemical Society. 134 (6), 2898-2901 (2012).</li></ol>

作者没有什么可透露的。

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 24,25的非自然氨基酸和工程正交平移元件 26,27,28 ,29。除了这些改进, 这里描述的系统提供了另一个能力, 一个非自然有机体的扩展遗传密码: 生物合成的非自然氨基酸。这种能力使非自然系统更独立作为一个有机体与扩展的基因保留。此外, 巴胺对这种方法所使用的细菌菌株有适度的毒性, 而在3毫米大巴中, 细胞生长明显减少, 导致蛋白质产量低。采用直接并网系统, 降低了毒性, 提高了蛋白质的产量。
aaRS 突变体用于小巴基因的融合具有适度的效率和保真度, 并在低浓度的巴胺中加入 Tyr。18因此, 多巴在细菌细胞中的生物合成应足够有效, 以防止 Tyr 合并。从邻苯二酚、氨和丙酮酸的 TPL 生物合成是一个可逆的反应, 高浓度的起始材料需要有效的生物合成。然而, 邻苯二酚不能使用超过10毫米, 因为它是有毒的细菌菌株在本议定书中使用。在这项协议中, 数码广播的浓度也很重要。多巴在转化为 l-dopaquinone 时表现出其毒性, 并在生长培养基中加入多巴, 以降低氧化速率。我们观察到, 当µM 浓度超过300时, 细胞生长下降。因此, 为了对巴胺进行最佳生物合成和合并, 应在本议定书所述的适当浓度下使用培养基中的成分 (特别是邻苯二酚和德勤)。
该协议还描述多巴多巴蛋白在特定地点蛋白共轭中的应用。多巴氧化产生的 l-dopaquinone 有效地与一个与生化探针相连的紧张炔烃反应。这种 cycloaddition 反应通常比亲核加法反应更快的胺或硫醇。然而, 多巴的氧化作用应在含多巴的蛋白质和紧张的炔烃的存在下进行, 以最大限度地减少蛋白质中亲核试剂的亲核增加。因此, 这个反应的试剂的添加顺序是至关重要的。
许多非自然氨基酸可用于蛋白质共轭, 其中一些能达到快速反应速率和优良的共轭效率30,31,32,33。这些氨基酸含有双正交功能基团, 如叠氮化合物、炔烃、紧张的烯烃或炔烃和 tetrazines。虽然它们对于特定于站点的蛋白质共轭是有用的, 但其中许多是昂贵的或商业上无法进入的, 这限制了它们的应用, 尤其是那些需要大规模蛋白质的产品。因此, 从廉价的起始材料中多巴的生物合成和它的直接结合将是有用的制药和工业应用需要的大规模突变蛋白含有生化探针或药物 (抗体药物共轭, ADC)。

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。

<table><tbody><tr><td>&lt;strong&gt;1. Plasmid Construction&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Plasmid pBAD-dual-TPL-GFP-E90TAG</td><td></td><td></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><td>Plasmid pEvol-DHPRS2</td><td></td><td></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><td>DH10&amp;beta;</td><td>Invitrogen</td><td>C6400-03</td><td>Expression Host</td></tr><tr><td>Plasmid Mini-prep kit</td><td>Nucleogen</td><td>5112</td><td>200/pack</td></tr><tr><td>Agarose</td><td>Intron biotechnology</td><td>32034</td><td>500 g</td></tr><tr><td>Ethidium bromide</td><td>Alfa Aesar</td><td>L07482</td><td>1 g</td></tr><tr><td>LB Broth</td><td>BD Difco</td><td>244620</td><td>500 g</td></tr><tr><td>&lt;strong&gt;2. Culture preparation&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>&lt;strong&gt;2.1) Electroporation&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Micro pulser&amp;nbsp;</td><td>BIO-RAD</td><td>165-2100</td><td></td></tr><tr><td>Micro pulser cuvette</td><td>BIO-RAD</td><td>165-2089</td><td>0.1 cm electrode gap, pkg. of 50</td></tr><tr><td>Ampicillin Sodium</td><td>Wako</td><td>018-10372</td><td>25 g</td></tr><tr><td>Chloramphenicol</td><td>Alfa Aesar</td><td>B20841</td><td>25 g</td></tr><tr><td>Agar</td><td>SAMCHUN</td><td>214230</td><td>500 g</td></tr><tr><td>SOC medium</td><td>Sigma</td><td>S1797</td><td>100 mL</td></tr><tr><td>&lt;strong&gt;3. Expression and Purification of GFP-E90DOPA by biosynthetic system&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>&lt;strong&gt;3.1 Expression of GFP-E90DOPA by biosynthetic system&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>L(+)-Arabinose, 99%</td><td>Acros</td><td>104981000</td><td>100 g</td></tr><tr><td>Pyrocatechol, 99%</td><td>SAMCHUN</td><td>P1387</td><td>25 g</td></tr><tr><td>Ammonium sulfate, 99%</td><td>SAMCHUN</td><td>A0943</td><td>500 g</td></tr><tr><td>pyruvic acid, 98%</td><td>Alfa Aesar</td><td>A13875</td><td>100 g</td></tr><tr><td>Sodium phosphate dibasic, anhydrous, 99%</td><td>SAMCHUN</td><td>S0891</td><td>1 kg</td></tr><tr><td>Potassium phophate, monobasic, 99%</td><td>SAMCHUN</td><td>P1127</td><td>1 kg</td></tr><tr><td>Magnesium sulfate, anhydrous, 99%</td><td>SAMCHUN</td><td>M0146</td><td>1 kg</td></tr><tr><td>D(+)-Glucose, anhydrous, 99%</td><td>SAMCHUN</td><td>D0092</td><td>500 g</td></tr><tr><td>Glycerol, 99%</td><td>SAMCHUN</td><td>G0269</td><td>1 kg</td></tr><tr><td>Trace metal mix a5 with co</td><td>Sigma</td><td>92949</td><td>25 mL</td></tr><tr><td>L-Proline, 99%</td><td>SAMCHUN</td><td>P1257</td><td>25 g</td></tr><tr><td>L-Phenylalanine, 98.5%</td><td>SAMCHUN</td><td>P1982</td><td>25 g</td></tr><tr><td>L-Tryptophane</td><td>JUNSEI</td><td>49550-0310</td><td>25 g</td></tr><tr><td>L-Arginine, 98%</td><td>SAMCHUN</td><td>A1149</td><td>25 g</td></tr><tr><td>L-Glutamine, 98%</td><td>JUNSEI</td><td>27340-0310</td><td>25 g</td></tr><tr><td>L-Asparagine monohydrate, 99%</td><td>SAMCHUN</td><td>A1198</td><td>25 g</td></tr><tr><td>L-Methionine</td><td>JUNSEI</td><td>73190-0410</td><td>25 g</td></tr><tr><td>L-Histidine hydrochloride monohydrate, 99%</td><td>SAMCHUN</td><td>H0604</td><td>25 g</td></tr><tr><td>L-Threonine, 99%</td><td>SAMCHUN</td><td>T2938</td><td>25 g</td></tr><tr><td>L-Leucine</td><td>JUNSEI</td><td>87070-0310</td><td>25 g</td></tr><tr><td>Glycine, 99%</td><td>SAMCHUN</td><td>G0286</td><td>25 g</td></tr><tr><td>L-Glutamic acid, 99%</td><td>SAMCHUN</td><td>G0233</td><td>25 g</td></tr><tr><td>L-Alanine, 99%</td><td>SAMCHUN</td><td>A1543</td><td>25 g</td></tr><tr><td>L-Isoleucine, 99%</td><td>SAMCHUN</td><td>I1049</td><td>25 g</td></tr><tr><td>L-Valine, 99%</td><td>SAMCHUN</td><td>V0088</td><td>25 g</td></tr><tr><td>L-Serine</td><td>SAMCHUN</td><td>S2447</td><td>25 g</td></tr><tr><td>L-Aspartic acid</td><td>SAMCHUN</td><td>A1205</td><td>25 g</td></tr><tr><td>L-Lysine monohydrochloride, 99%</td><td>SAMCHUN</td><td>L0592</td><td>25 g</td></tr><tr><td>&lt;strong&gt;3.2 Cell lysis&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Imidazole, 99%</td><td>SAMCHUN</td><td>I0578</td><td>1kg</td></tr><tr><td>Sodium phosphate monobasic, 98%</td><td>SAMCHUN</td><td>S0919</td><td>1 kg</td></tr><tr><td>Sodium Chloride, 99%</td><td>SAMCHUN</td><td>S2907</td><td>1 kg</td></tr><tr><td>Ultrasonic Processor - 150 microliters to 150 milliliters</td><td>SONIC &amp;amp; MATERIALS</td><td>VCX130</td><td></td></tr><tr><td>&lt;strong&gt;3.3 Ni-NTA Affinity Chromatography&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Ni-NTA resin</td><td>QIAGEN</td><td>30210</td><td>25 mL</td></tr><tr><td>Polypropylene column</td><td>QIAGEN</td><td>34924</td><td>50/pack, 1 mL capacity</td></tr><tr><td>&lt;strong&gt;4. Oligomerization of Purified GFP-E90DOPA&amp;nbsp;&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Sodium periodate, 99.8&amp;amp;</td><td>Acros</td><td>419610050</td><td>5 g</td></tr><tr><td>&lt;strong&gt;5. Conjugation of GFP-E90DOPA with an Alkyne Probe by Strain-Promoted Oxidation-Controlled Cyclooctyne&amp;ndash;1,2-Quinone Cycloaddition (SPOCQ)&amp;nbsp;&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Cy5.5-ADIBO&amp;nbsp;</td><td>FutureChem</td><td>FC-6119</td><td>1mg</td></tr><tr><td>&lt;strong&gt;6. Purification of Labeled GFP&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Amicon Ultra 0.5 mL Centrifugal Filters</td><td>MILLIPORE</td><td>UFC500396</td><td>96/pack, 500ul capacity</td></tr><tr><td>&lt;strong&gt;7. SDS-PAGE Analysis and Fluorescence Gel Scanning&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>1,4-Dithio-DL-threitol, DTT, 99.5 %</td><td>Sigma</td><td>10708984001</td><td>10 g</td></tr><tr><td>NuPAGE LDS Sample Buffer, 4X</td><td>Thermofisher</td><td>NP0007</td><td>10 mL</td></tr><tr><td>MES running buffer</td><td>Thermofisher</td><td>NP0002</td><td>500 mL</td></tr><tr><td>Nupage Novex 4-12% SDS PAGE gels</td><td>Thermofisher</td><td>NO0321</td><td>12 well</td></tr><tr><td>Coomassie Brilliant Blue R-250</td><td>Wako</td><td>031-17922</td><td>25 g</td></tr><tr><td>G:BOX Chemi Fluorescent &amp;amp; Chemiluminescent Imaging System</td><td>Syngene</td><td></td><td>G BOX Chemi XT4</td></tr><tr><td>&lt;strong&gt;8. MALDI-TOF MS analysis by Trypsin Digestion&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>&lt;strong&gt;8.1 Preparation of the digested peptide sample by trypsin digestion&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Tris(hydroxymethyl)aminomethane, 99%</td><td>SAMCHUN</td><td>T1351</td><td>500 g</td></tr><tr><td>Hydrochloric acid, 35~37%</td><td>SAMCHUN</td><td>H0256</td><td>500 mL</td></tr><tr><td>Dodecyl sulfate sodium salt, 85%</td><td>SAMCHUN</td><td>D1070</td><td>250 g</td></tr><tr><td>Iodoacetamide</td><td>Sigma</td><td>I6125</td><td>5 g</td></tr><tr><td>Trypsin Protease, MS Grade</td><td>Thermofisher</td><td>90057</td><td>5 x 20 &amp;micro;g/pack</td></tr><tr><td>C-18 spin columns</td><td>Thermofisher</td><td>89870</td><td>25/pack, 200 &amp;micro;L capacity</td></tr><tr><td>&lt;strong&gt;8.2 Analysis of the digested peptide by MALDI-TOF&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Acetonitirile, 99.5%</td><td>SAMCHUN</td><td>A0125</td><td>500 mL</td></tr><tr><td>&amp;alpha;-Cyano-4-hydroxycinnamic acid</td><td>Sigma</td><td>C2020</td><td>10 g</td></tr><tr><td>Trifluoroacetic acid, 99%</td><td>SAMCHUN</td><td>T1666</td><td>100 g</td></tr><tr><td>MTP 384 target plate ground steel BC targets</td><td>Bruker</td><td>8280784</td><td></td></tr><tr><td>Bruker Autoflex Speed MALDI-TOF mass spectrometer</td><td>Bruker</td><td></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。本实验所用的生长培养基中含有25毫米氨和100毫米丙酮酸, 其浓度变化为邻苯二酚 (0-10 毫米)。邻苯二酚的浓度对多巴生物合成是至关重要的, 而氨和丙酮酸浓度分别超过25毫米和100毫米, 并没有影响巴多巴的生物合成和加入。邻苯二酚的最佳浓度为10毫米, 浓度超过10毫米, 显著降低细菌细胞生长, 其细胞毒性。在这种最佳条件下, TPL 从其起始材料中 biosynthesized 了巴比, 而 biosynthesized 巴则直接加入了 GFP。用 SDS (图 2A) 纯化和分析了表达的突变体 GFP (GFP-E90DOPA)。全长的 GFP 被纯化, 多巴合并由 MALDI 的 MS 分析证实 (图 2B)。用胰蛋白酶消化蛋白, 对多巴多肽片段进行分析, 结果表明多巴与无可检测 Tyr 或其它天然氨基酸结合。第三方 biosynthesized 的并网效率与3毫米巴的结合效率相似, 因为它的细胞毒性19, 是巴比的最大浓度。虽然10毫米邻苯二酚显示中等毒性, 细胞密度在 16 h 以后文化时间是三-到四重在高于3毫米多巴的存在。生物合成纯化后的蛋白质产量比实验使用3毫米多巴的纯度高三倍 (图 3)。
含有巴胺的突变型 GFP 用于蛋白质共轭。多巴可通过轻度氧化剂氧化成 l-dopaquinone, 亲醌与炔烃和亲核试剂 (如硫醇和胺) 的紧张反应, 可用于 bioconjugation (图 4)。GFP-E90DOPA 的 SPOCQ cycloaddition 与高碘酸钠反应, 其次是治疗与 Cy5.5 ADIBO22。通过 SDS 页和荧光成像 (图 5A) 对 SPOCQ 反应进行了分析。通过对高碘酸钠和 Cy5.5 ADIBO 的治疗, 观察到强烈的荧光带, 而无荧光带显示在与 GFP 或没有高碘酸钠治疗的控制反应中, 确认其效率和特异性共轭反应与基因合并的多巴。此外, 含有多巴的 GFP 被用于蛋白质齐聚。l-dopaquinone 反应与赖氨酸或胱氨酸在同一蛋白质, 导致寡聚蛋白。GFP-E90DOPA 经高碘酸钠治疗, 并进行了48小时的齐聚反应, 并与对照样品进行了比较, 并与不含高碘酸钠治疗的控制样本进行了对比分析 (图 5B)。分析表明, 在高碘酸盐处理的样品中, 具有相同间隔蛋白质带的明显的齐聚型, 而对照样品则显示完整的蛋白质, 没有任何其它带。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/58383/58383fig1.jpg"> 图 1: 直接纳入邻苯二酚、丙酮酸和氨的 biosynthesized.heterologously 是由邻苯二酚、丙酮酸和氨在生长培养基中表达的 biosynthesized。进化的 tRNA/aaRS 对多巴是共同表达, 以合并 biosynthesized 多巴入靶蛋白, GFP。TPL 基因被放置在本构启动子之下, 以便在诱导 GFP 表达之前开始巴比生物合成。<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58383/58383fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/58383/58383fig2.jpg"> 图 2: 对设计的生物合成系统表达的 GFP-E90DOPA 进行了 MALDI 分析.(A) GFP-E90DOPA 在10毫米邻苯二酚、100毫米丙酮酸盐和25毫米氨的存在下被设计的生物合成系统表达, 然后用 Ni-NTA 亲和层析纯化。并对 GFP 进行了比较分析。样品由二-三 4%-12% SDS 页分离, 并且凝胶被染了考马斯明亮的蓝色 R-250。(B) MALDI 对含有多巴的多巴肽片段进行 GFP-E90DOPA 胰蛋白酶消化的分析。GFP 的肽片段含有 86-96 (SAMPEGYVQER) 的残留物。从 GFP-E90DOPA 的肽片段中, 用多巴取代了 GFP 在片段中的谷氨酸。<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58383/58383fig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/58383/58383fig3.jpg"> 图 3: 比较 biosynthesized 巴与一巴的一般遗传合并.这些面板显示的 GFP-E90DOPA 表达的设计生物合成系统和基因成立的存在3毫米多巴, 没有额外的丙酮酸, 氨, 和邻苯二酚。(A) 从纯化的 GFP-E90DOPA 中获得蛋白质的产量, (B) 在诱导后16小时测量细胞密度。<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58383/58383fig3large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/58383/58383fig4.jpg"> 图 4: GFP-E90DOPA bioconjugation 的反应方案.GFP-E90DOPA 被氧化成 dopaquinone。dopaquinone 蛋白与张力炔烃的反应达到特定部位的蛋白质标记。长时间 (48 小时) 高浓度 dopaquinone 蛋白的孵化导致蛋白质的自共轭产生蛋白质寡聚物。<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58383/58383fig4large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/58383/58383fig5.jpg"> 图 5: Bioconjugation 含有几巴的突变蛋白.(a) 多巴多巴蛋白与紧张的炔烃、ADIBO Cy5.5 的 SPOCQ 反应。用高碘酸钠治疗 GFP 和 GFP-E90DOPA, 并与 ADIBO Cy5.5 反应60分钟。用 SDS 页对反应进行了分析, 显示了考马斯染色 (上) 和荧光 (下) 图像。(B) GFP-E90DOPA 的齐聚。用高碘酸钠治疗多巴含有的 GFP, 孵化48小时。用 SDS 页分析了反应混合物。<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58383/58383fig5large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>

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 ...

genetic-incorporation-biosynthesized-l-dihydroxyphenylalanine-dopa

Nanyang Technological University

Research

JoVE Journal

Biochemistry

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

7.8K 次观看.  Sogang University. 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。

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

Efficient and Site-specific Antibody Labeling by Strain-promoted Azide-alkyne Cycloaddition

There are currently many chemical tools available to introduce chemical probes into proteins to study their structure and function. A useful method is protein conjugation by genetically introducing an unnatural amino acid containing a bioorthogonal functional group. This report describes a detailed protocol for site-specific antibody conjugation. The protocol includes experimental details for the genetic incorporation of an azide-containing amino acid, and the conjugation reaction by strain-promoted azide-alkyne cycloaddition (SPAAC). This strain-promoted reaction proceeds by simple mixing of the reacting molecules at physiological pH and temperature, and does not require additional reagents such as copper(I) ions and copper-chelating ligands. Therefore, this method would be useful for general protein conjugation and development of antibody drug conjugates (ADCs).

Here, we present a protocol to site-specifically introduce chemical probes into an antibody fragment by genetically incorporating an azide-containing amino acid, and subsequently coupling the azide with a chemical probe by strain-promoted azide-alkyne cycloaddition (SPAAC).

高效抗体标记由应变叠氮推广 - 炔环加成特定站点

There are currently many chemical tools available to introduce chemical probes into proteins to study their structure and function. A useful method is ...

科研

生物化学

Optimizing the Genetic Incorporation of Chemical Probes into GPCRs for Photo-crosslinking Mapping and Bioorthogonal Chemistry in Live Mammalian Cells

The genetic incorporation of non-canonical amino acids (ncAAs) via amber stop codon suppression is a powerful technique to install artificial probes and reactive moieties onto proteins directly in the live cell. Each ncAA is incorporated by a dedicated orthogonal suppressor-tRNA/amino-acyl-tRNA-synthetase (AARS) pair that is imported into the host organism. The incorporation efficiency of different ncAAs can greatly differ, and be unsatisfactory in some cases. Orthogonal pairs can be improved by manipulating either the AARS or the tRNA. However, directed evolution of tRNA or AARS using large libraries and dead/alive selection methods are not feasible in mammalian cells. Here, a facile and robust fluorescence-based assay to evaluate the efficiency of orthogonal pairs in mammalian cells is presented. The assay allows screening tens to hundreds of AARS/tRNA variants with a moderate effort and within a reasonable time. Use of this assay to generate new tRNAs that significantly improve the efficiency of the pyrrolysine orthogonal system is described, along with the application of ncAAs to the study of G-protein coupled receptors (GPCRs), which are challenging objects for ncAA mutagenesis. First, by systematically incorporating a photo-crosslinking ncAA throughout the extracellular surface of a receptor, binding sites of different ligands on the intact receptor are mapped directly in the live cell. Second, by incorporating last-generation ncAAs into a GPCR, ultrafast catalyst-free receptor labeling with a fluorescent dye is demonstrated, which exploits bioorthogonal strain-promoted inverse Diels Alder cycloaddition (SPIEDAC) on the live cell. As ncAAs can be generally applied to any protein independently on its size, the method is of general interest for a number of applications. In addition, ncAA incorporation does not require any special equipment and is easily performed in standard biochemistry labs.

A facile fluorescence assay is presented to evaluate the efficiency of amino-acyl-tRNA-synthetase/tRNA pairs incorporating non-canonical amino-acids (ncAAs) into proteins expressed in mammalian cells. The application of ncAAs to study G-protein coupled receptors (GPCRs) is described, including photo-crosslinking mapping of binding sites and bioorthogonal GPCR labeling on live cells.

生物哺乳动物细胞光交联图谱和 Bioorthogonal 化学中化学探针的遗传受体优化

The genetic incorporation of non-canonical amino acids (ncAAs) via amber stop codon suppression is a powerful technique to install artificial probes and ...

化学

Genetic Encoding of a Non-Canonical Amino Acid for the Generation of Antibody-Drug Conjugates Through a Fast Bioorthogonal Reaction

Antibody-drug conjugates (ADCs) used nowadays in clinical practice are mixtures of antibody molecules linked to a varying number of toxins at different positions. Preclinical studies have shown that the therapeutic index of these traditional ADCs can be improved by the site-specific linkage of toxins. However, current approaches to produce homogeneous ADCs have several limitations, such as low protein expression and slow reaction kinetics. In this protocol we describe how to set up an expression system to incorporate a cyclopropene derivative of lysine (CypK) into antibodies using genetic code expansion. This minimal bioorthogonal handle allows rapid conjugation of tetrazine derivatives through an inverse-demand Diels-Alder cycloaddition. The expression system here reported enables the facile production and purification of trastuzumab bearing CypK in each of the heavy chains. We explain how to link the antibody to the toxin monomethyl auristatin E and characterize the immunoconjugate by hydrophobic interaction chromatography and mass spectrometry. Finally, we describe assays to assess the stability in human serum of the dihydropyridazine linkage resulting from the conjugation and to test the selective cytotoxicity of the ADC for breast cancer cells with high levels of HER2 receptor.

Incorporating a cyclopropene derivative of lysine into antibodies allows the site-specific, rapid and efficient linkage of tetrazine-bearing molecules to generate antibody-drug conjugates.

用快速 Bioorthogonal 反应生成抗体-药物共轭物的非规范氨基酸的遗传编码

Antibody-drug conjugates (ADCs) used nowadays in clinical practice are mixtures of antibody molecules linked to a varying number of toxins at different ...

OaAEP1-Mediated Enzymatic Synthesis and Immobilization of Polymerized Protein for Single-Molecule Force Spectroscopy

Chemical and bio-conjugation techniques have been developed rapidly in recent years and allow the building of protein polymers. However, a controlled protein polymerization process is always a challenge. Here, we have developed an enzymatic methodology for constructing polymerized protein step by step in a rationally-controlled sequence. In this method, the C-terminus of a protein monomer is NGL for protein conjugation using OaAEP1 (Oldenlandia affinis asparaginyl endopeptidases) 1) while the N-terminus was a cleavable TEV (tobacco etch virus) cleavage site plus an L (ENLYFQ/GL) for temporary N-terminal protecting. Consequently, OaAEP1 was able to add only one protein monomer at a time, and then the TEV protease cleaved the N-terminus between Q and G to expose the NH2-Gly-Leu. Then the unit is ready for next OaAEP1 ligation. The engineered polyprotein is examined by unfolding individual protein domain using atomic force microscopy-based single-molecule force spectroscopy (AFM-SMFS). Therefore, this study provides a useful strategy for polyprotein engineering and immobilization.

Here, we present a protocol to conjugate protein monomer by enzymes forming protein polymer with a controlled sequence and immobilize it on the surface for single-molecule force spectroscopy studies.

OaAEP1介导酶合成和聚合蛋白固定，用于单分子力光谱

Chemical and bio-conjugation techniques have been developed rapidly in recent years and allow the building of protein polymers. However, a controlled ...

Engineering 'Golden' Fluorescence by Selective Pressure Incorporation of Non-canonical Amino Acids and Protein Analysis by Mass Spectrometry and Fluorescence

Fluorescent proteins are fundamental tools for the life sciences, in particular for fluorescence microscopy of living cells. While wild-type and engineered variants of the green fluorescent protein from Aequorea victoria (avGFP) as well as homologs from other species already cover large parts of the optical spectrum, a spectral gap remains in the near-infrared region, for which avGFP-based fluorophores are not available. Red-shifted fluorescent protein (FP) variants would substantially expand the toolkit for spectral unmixing of multiple molecular species, but the naturally occurring red-shifted FPs derived from corals or sea anemones have lower fluorescence quantum yield and inferior photo-stability compared to the avGFP variants. Further manipulation and possible expansion of the chromophore&#39;s conjugated system towards the far-red spectral region is also limited by the repertoire of 20 canonical amino acids prescribed by the genetic code. To overcome these limitations, synthetic biology can achieve further spectral red-shifting via insertion of non-canonical amino acids into the chromophore triad. We describe the application of SPI to engineer avGFP variants with novel spectral properties. Protein expression is performed in a tryptophan-auxotrophic E. coli strain and by supplementing growth media with suitable indole precursors. Inside the cells, these precursors are converted to the corresponding tryptophan analogs and incorporated into proteins by the ribosomal machinery in response to UGG codons. The replacement of Trp-66 in the enhanced &#34;cyan&#34; variant of avGFP (ECFP) by an electron-donating 4-aminotryptophan results in GdFP featuring a 108 nm Stokes shift and a strongly red-shifted emission maximum (574 nm), while being thermodynamically more stable than its predecessor ECFP. Residue-specific incorporation of the non-canonical amino acid is analyzed by mass spectrometry. The spectroscopic properties of GdFP are characterized by time-resolved fluorescence spectroscopy as one of the valuable applications of genetically encoded FPs in life sciences.

Synthetic biology enables the engineering of proteins with unprecedented properties using the co-translational insertion of non-canonical amino acids. Here, we presented how a spectrally red-shifted variant of a GFP-type fluorophore with novel fluorescence spectroscopic properties, termed &#34;gold&#34; fluorescent protein (GdFP), is produced in E. coli via selective pressure incorporation (SPI).

用质谱和荧光法研究非规范氨基酸和蛋白质分析的选择性压力下工程 "金" 荧光

Fluorescent proteins are fundamental tools for the life sciences, in particular for fluorescence microscopy of living cells. While wild-type and ...

生物工程

A Facile Protocol to Generate Site-Specifically Acetylated Proteins in Escherichia Coli

Post-translational modifications that occur at specific positions of proteins have been shown to play important roles in a variety of cellular processes. Among them, reversible lysine acetylation is one of the most widely distributed in all domains of life. Although numerous mass spectrometry-based acetylome studies have been performed, further characterization of these putative acetylation targets has been limited. One possible reason is that it is difficult to generate purely acetylated proteins at desired positions by most classic biochemical approaches. To overcome this challenge, the genetic code expansion technique has been applied to use the pair of an engineered pyrrolysyl-tRNA synthetase variant, and its cognate tRNA from Methanosarcinaceae species, to direct the cotranslational incorporation of acetyllysine at the specific site in the protein of interest. After first application in the study of histone acetylation, this approach has facilitated acetylation studies on a variety of proteins. In this work, we demonstrated a facile protocol to produce site-specifically acetylated proteins by using the model bacterium Escherichia coli as the host. Malate dehydrogenase was used as a demonstration example in this work.

A Facile Protocol to Generate Site-Specifically Acetylated Proteins in Escherichia Coli

Genetic code expansion serves as a powerful tool to study a wide range of biological processes, including protein acetylation. Here we demonstrate a facile protocol to exploit this technique for generating homogeneously acetylated proteins at specific sites in Escherichia coli cells.

在大肠杆菌中生成特异的乙酰蛋白的简易协议

Post-translational modifications that occur at specific positions of proteins have been shown to play important roles in a variety of cellular processes. ...

免疫与感染

Homogeneous Glycoconjugate Produced by Combined Unnatural Amino Acid Incorporation and Click-Chemistry for Vaccine Purposes

Genetic code expansion is a powerful tool to introduce unnatural amino acids (UAAs) into proteins to modify their characteristics, to study or create new protein functions or to have access to protein conjugates. Stop codon suppression, in particular amber codon suppression, has emerged as the most popular method to genetically introduce UAAs at defined positions. This methodology is herein applied to the preparation of a carrier protein containing an UAA harboring a bioorthogonal functional group. This reactive handle can next be used to specifically and efficiently graft a synthetic oligosaccharide hapten to provide a homogeneous glycoconjugate vaccine. The protocol is limited to the synthesis of glycoconjugates in a 1:1 carbohydrate hapten/carrier protein ratio but amenable to numerous pairs of biorthogonal functional groups. Glycococonjugate vaccine homogeneity is an important criterion to ensure complete physico-chemical characterization, thereby, satisfying more and more demanding drug regulatory agency recommendations, a criterion which is unmet by classical conjugation strategies. Moreover, this protocol makes it possible to finely tune the structure of the actual conjugate vaccine, giving rise to tools to address structure-immunogenicity relationships.

Genetic code expansion is applied for the introduction of an unnatural amino acid bearing a biorthogonal functional group on a carrier protein at a defined site. The biorthogonal function is further used for the site-selective coupling of a carbohydrate antigen to provide a homogeneous glycoconjugate vaccine.

联合非自然氨基酸合并和点击化学为疫苗目的生产的同质糖共酶

Genetic code expansion is a powerful tool to introduce unnatural amino acids (UAAs) into proteins to modify their characteristics, to study or create new ...

Residue-Specific Exchange of Proline by Proline Analogs in Fluorescent Proteins: How "Molecular Surgery" of the Backbone Affects Folding and Stability

Replacement of proline (Pro) residues in proteins by the traditional site-directed mutagenesis by any of the remaining 19 canonical amino acids is often detrimental to protein folding and, in particular, chromophore maturation in green fluorescent proteins and related variants. A reasonable alternative is to manipulate the translation of the protein so that all Pro residues are replaced residue-specifically by analogs, a method known as selective pressure incorporation (SPI). The built-in chemical modifications can be used as a kind of &#34;molecular surgery&#34; to finely dissect measurable changes or even rationally manipulate different protein properties. Here, the study demonstrates the usefulness of the SPI method to study the role of prolines in the organization of the typical &#946;-barrel structure of spectral variants of the green fluorescent protein (GFP) with 10-15 prolines in their sequence: enhanced green fluorescent protein (EGFP), NowGFP, and KillerOrange. Pro residues are present in connecting sections between individual &#946;-strands and constitute the closing lids of the barrel scaffold, thus being responsible for insulation of the chromophore from water, i.e., fluorescence properties. Selective pressure incorporation experiments with (4R)-fluoroproline (R-Flp), (4S)-fluoroproline (S-Flp), 4,4-difluoroproline (Dfp), and 3,4-dehydroproline (Dhp) were performed using a proline-auxotrophic E. coli strain as expression host. We found that fluorescent proteins with S-Flp and Dhp are active (i.e., fluorescent), while the other two analogs (Dfp and R-Flp) produced dysfunctional, misfolded proteins. Inspection of UV-Vis absorption and fluorescence emission profiles showed few characteristic alterations in the proteins containing Pro analogs. Examination of the folding kinetic profiles in EGFP variants showed an accelerated refolding process in the presence of S-Flp, while the process was similar to wild-type in the protein containing Dhp. This study showcases the capacity of the SPI method to produce subtle modifications of protein residues at an atomic level (&#34;molecular surgery&#34;), which can be adopted for the study of other proteins of interest. It illustrates the outcomes of proline replacements with close chemical analogs on the folding and spectroscopic properties in the class of &#946;-barrel fluorescent proteins.

To overcome the limitations of classical site-directed mutagenesis, proline analogs with specific modifications were incorporated into several fluorescent proteins. We show how the replacement of hydrogen by fluorine or of the single by double bonds in proline residues ("molecular surgery") affects fundamental protein properties, including their folding and interaction with light.

荧光蛋白中脯氨酸类似物对脯氨酸的残基特异换:骨架的"分子手术"如何影响折叠和稳定性

Replacement of proline (Pro) residues in proteins by the traditional site-directed mutagenesis by any of the remaining 19 canonical amino acids is often ...

Genetically-encoded Molecular Probes to Study G Protein-coupled Receptors

To facilitate structural and dynamic studies of G protein-coupled receptor (GPCR) signaling complexes, new approaches are required to introduce informative probes or labels into expressed receptors that do not perturb receptor function. We used amber codon suppression technology to genetically-encode the unnatural amino acid, p-azido-L-phenylalanine (azF) at various targeted positions in GPCRs heterologously expressed in mammalian cells. The versatility of the azido group is illustrated here in different applications to study GPCRs in their native cellular environment or under detergent solubilized conditions. First, we demonstrate a cell-based targeted photocrosslinking technology to identify the residues in the ligand-binding pocket of GPCR where a tritium-labeled small-molecule ligand is crosslinked to a genetically-encoded azido amino acid. We then demonstrate site-specific modification of GPCRs by the bioorthogonal Staudinger-Bertozzi ligation reaction that targets the azido group using phosphine derivatives. We discuss a general strategy for targeted peptide-epitope tagging of expressed membrane proteins in-culture and its detection using a whole-cell-based ELISA approach. Finally, we show that azF-GPCRs can be selectively tagged with fluorescent probes. The methodologies discussed are general, in that they can in principle be applied to any amino acid position in any expressed GPCR to interrogate active signaling complexes.

We genetically-encode the unnatural amino acid, p-azido-L-phenylalanine at various targeted positions in GPCRs and show the versatility of the azido group in different applications. These include a targeted photocrosslinking technology to identify residues in the ligand-binding pocket of a GPCR, and site-specific bioorthogonal modification of GPCRs with a peptide-epitope tag or fluorescent probe.

基因编码的分子探针来研究G蛋白偶联受体

To facilitate structural and dynamic studies of G protein-coupled receptor (GPCR) signaling complexes, new approaches are required to introduce ...

生物学

Functional Complementation Analysis (FCA): A Laboratory Exercise Designed and Implemented to Supplement the Teaching of Biochemical Pathways

Functional complementation assay (FCA) is an in vivo assay that is widely used to elucidate the function/role of genes/enzymes. This technique is very common in biochemistry, genetics and many other disciplines. A comprehensive overview of the technique to supplement the teaching of biochemical pathways pertaining to amino acids, peptidoglycan and the bacterial stringent response is reported in this manuscript. Two cDNAs from the model plant organism Arabidopsis thaliana that are involved in the metabolism of lysine (L,L-diaminopimelate aminotransferase (dapL) and tyrosine aminotransferase (tyrB) involved in the metabolism of tyrosine and phenylalanine are highlighted. In addition, the bacterial peptidoglycan anabolic pathway is highlighted through the analysis of the UDP-N-acetylmuramoyl-L-alanyl-D-glutamate-meso-2,6-diaminopimelate ligase (murE) gene from the bacterium Verrucomicrobium spinosum involved in the cross-linking of peptidoglycan. The bacterial stringent response is also reported through the analysis of the rsh (relA/spoT homolog) bifunctional gene responsible for a hyper-mucoid phenotype in the bacterium Novosphingobium sp. Four examples of FCA are presented. The video will focus on three of them, namely lysine, peptidoglycan and the stringent response.

Functional Complementation Analysis FCA: A Laboratory Exercise Designed and Implemented to Supplement the Teaching of Biochemical Pathways

The validation of enzymatic activities involved in biochemical pathways can be elucidated using functional complementation analysis (FCA). Described in this manuscript is the FCA assay demonstrating the enzymatic activity of enzymes involved in the metabolism of amino acids, bacterial stringent response and bacterial peptidoglycan biosynthesis.

功能互补分析（FCA）:实验室练习设计和实施，以补充生化途径的教学

Functional complementation assay (FCA) is an in vivo assay that is widely used to elucidate the function/role of genes/enzymes. This technique is very ...