Мы хотели бы поблагодарить д-ра Уэсли Вана за то, что он заложил основу для этого протокола и его ценного наставничества. Эта работа была частично поддержана Национальными институтами здравоохранения (Grants R01GM127575 и R01GM121584) и Фондом Уэлча (Grant A-1715).

1. Плазмидная конструкция
<ol><li>Начните с принятия решения, какой белок гистон будет ацетилирован и на котором лизин сайта. Мутировать сайт к янтарной остановки кодон (TAG) с помощью сайта направлены мутагенез. ПРИМЕЧАНИЕ: Есть четыре ранее разработанных плазмидов, используемых для выра...

<ol>
	<li>Srinivasan, G., James, C. M., Krzycki, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pyrrolysine+encoded+by+UAG+in+Archaea:+Charging+of+a+UAG-Decoding+specialized+tRNA.">Pyrrolysine encoded by UAG in Archaea: Charging of a UAG-Decoding specialized tRNA.</a> Science. 296 (5572), 1459-1462 (2002).</li><li>Wan, W., Tharp, J. M., Liu, W. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pyrrolysyl-tRNA+synthetase:+an+ordinary+enzyme+but+an+outstanding+genetic+code+expansion+tool.">Pyrrolysyl-tRNA synthetase: an ordinary enzyme but an outstanding genetic code expansion tool.</a> Biochimica Et Biophysica Acta. 1844 (6), 1059-1070 (2014).</li><li>Umehara, 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=N-Acetyl+lysyl-tRNA+synthetases+evolved+by+a+CcdB-based+selection+possess+N-acetyl+lysine+specificity+in+vitro+and+in+vivo.">N-Acetyl lysyl-tRNA synthetases evolved by a CcdB-based selection possess N-acetyl lysine specificity in vitro and in vivo.</a> FEBS Letters. 586 (6), 729-733 (2012).</li><li>Hsu, W. W., Wu, B., Liu, W. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sirtuins+1+and+2+are+universal+histone+deacetylases.">Sirtuins 1 and 2 are universal histone deacetylases.</a> ACS Chemical Biology. 11 (3), 792-799 (2016).</li><li>Wang, W. W., Zeng, Y., Wu, B., Deiters, A., Liu, W. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+chemical+biology+approach+to+reveal+Sirt6-targeted+histone+H3+sites+in+nucleosomes.">A chemical biology approach to reveal Sirt6-targeted histone H3 sites in nucleosomes.</a> ACS Chemical Biology. 11 (7), 1973-1981 (2016).</li><li>Wang, W. W., 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+click+chemistry+approach+reveals+the+chromatin-dependent+histone+H3K36+deacylase+nature+of+SIRT7.">A click chemistry approach reveals the chromatin-dependent histone H3K36 deacylase nature of SIRT7.</a> Journal of the American Chemical Society. 141 (6), 2462-2473 (2019).</li><li>Liu, H., Naismith, 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=An+efficient+one-step+site-directed+deletion,+insertion,+single+and+multiple-site+plasmid+mutagenesis+protocol.">An efficient one-step site-directed deletion, insertion, single and multiple-site plasmid mutagenesis protocol.</a> BMC Biotechnology. 8 (1), 91 (2008).</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>Gallego-Jara, J., &#201;cija Conesa, A., de Diego Puente, T., Lozano Terol, G., C&#225;novas D&#237;az, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+CobB+kinetics+and+inhibition+by+nicotinamide.">Characterization of CobB kinetics and inhibition by nicotinamide.</a> PLoS One. 12 (12), 0189689 (2017).</li><li>Lowary, P. T., Widom, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+DNA+sequence+rules+for+high+affinity+binding+to+histone+octamer+and+sequence-directed+nucleosome+positioning.">New DNA sequence rules for high affinity binding to histone octamer and sequence-directed nucleosome positioning.</a> Journal of Molecular Biology. 276 (1), 19-42 (1998).</li><li>Zhao, B., 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=The+molecular+basis+of+tight+nuclear+tethering+and+inactivation+of+cGAS.">The molecular basis of tight nuclear tethering and inactivation of cGAS.</a> Nature. 587 (7835), 673-677 (2020).</li><li>Burdette, D. L., Vance, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=STING+and+the+innate+immune+response+to+nucleic+acids+in+the+cytosol.">STING and the innate immune response to nucleic acids in the cytosol.</a> Nature Immunology. 14 (1), 19-26 (2013).</li><li>Barber, G. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Innate+immune+DNA+sensing+pathways:+STING,+AIMII+and+the+regulation+of+interferon+production+and+inflammatory+responses.">Innate immune DNA sensing pathways: STING, AIMII and the regulation of interferon production and inflammatory responses.</a> Current Opinion in Immunology. 23 (1), 10-20 (2011).</li><li>Ablasser, A., 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=cGAS+produces+a+2'-5'-linked+cyclic+dinucleotide+second+messenger+that+activates+STING.">cGAS produces a 2'-5'-linked cyclic dinucleotide second messenger that activates STING.</a> Nature. 498 (7454), 380-384 (2013).</li><li>Gao, P., 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=Cyclic+[G(2',5')pA(3',5')p]+is+the+metazoan+second+messenger+produced+by+DNA-activated+cyclic+GMP-AMP+synthase.">Cyclic [G(2',5')pA(3',5')p] is the metazoan second messenger produced by DNA-activated cyclic GMP-AMP synthase.</a> Cell. 153 (5), 1094-1107 (2013).</li><li>Zhao, B., 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+conserved+PLPLRT/SD+motif+of+STING+mediates+the+recruitment+and+activation+of+TBK1.">A conserved PLPLRT/SD motif of STING mediates the recruitment and activation of TBK1.</a> Nature. 569 (7758), 718-722 (2019).</li></ol>

Важно следовать этому протоколу во всех деталях во время эксперимента. Нуклеосомы не очень стабильны, и при определении этого протокола было допущено много проб и ошибок. Это ключ к удалению осадков на каждом шагу (или при наблюдении), потому что частицы могут легко вмешиваться в процес?...

Мы использовали PylRS и tRNAPyl для синтеза и сборки восстановленных мононуклеосом с конкретными ацетилированными гистонами. PylRS оказался бесценным в качестве инструмента расширения генетического кода для производства белков с пост-трансляционными модификациями (PTMs) и был генетически эволюционировал, чтобы включить около 200 различных ncAAs. PylRS включает в себя на янтарной остановке кодон, устраняя конкуренцию с другими аминокислотами во время перевода. PylRS был впервые обнаружен в метамфетаминовых археев, и с тех пор был использован в химической биологии для включения новых реактивных химическихгрупп в белки 1,2.
MmAcKRS1 был создан из метаносаркина mazei PylRS и часто используется в нашей лаборатории для синтеза ацетилированныхбелков 3,4,5,6. MmAcKRS1 поставляет AcK на место мутации янтаря в мРНК выбора во время перевода в Escherichia coli. Этот метод ранее был использован для включения AcK на K4, K9, K14, K18, K23, K27, K36, K56, K64, и K79 histone H3 лизин сайтов для изучения деятельности Sirtuin 1, 2, 6, и 7 на ацетилированных мононуклеозом4,5,6. Здесь мы подробно этот метод, чтобы выразить ацетилированные гистоны и восстановить ацетилированные нуклеосомы.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.5 M TE Buffer</td>
			<td>NA</td>
			<td>NA</td>
			<td>0.5 M NaCl, 20 mM Tris, 1 mM EDTA, pH 7.8</td>
		</tr>
		<tr>
			<td>1 M TE Buffer</td>
			<td>NA</td>
			<td>NA</td>
			<td>1 M NaCl, 20 mM Tris, 1 mM EDTA, pH 7.8</td>
		</tr>
		<tr>
			<td>100x TE Buffer</td>
			<td>NA</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>2 M TE Buffer</td>
			<td>NA</td>
			<td>NA</td>
			<td>2 M NaCl, 20 mM Tris, 1 mM EDTA, pH 7.8</td>
		</tr>
		<tr>
			<td>20 mM TE Buffer</td>
			<td>NA</td>
			<td>NA</td>
			<td>20 mM NaCl, 20 mM Tris, 1 mM EDTA, pH 7.8</td>
		</tr>
		<tr>
			<td>6 M GuHCl</td>
			<td></td>
			<td></td>
			<td>6M guanidinium chloride, 20 mM Tris, 500 mM NaCl, pH 8.0</td>
		</tr>
		<tr>
			<td>Acetyllysine</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Column Wash Buffer</td>
			<td>NA</td>
			<td>NA</td>
			<td>6 M urea, 500 mM NaCl, 20 mM Tris, 20 mM imidazole pH 7.8</td>
		</tr>
		<tr>
			<td>Elution Buffer</td>
			<td>NA</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>Fisherbrand&#160;Variable-Flow Chemical Transfer Pump</td>
			<td>Fischer Scientific</td>
			<td>15-077-67</td>
			<td></td>
		</tr>
		<tr>
			<td>His-TEV protease</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Histone Lysis Buffer</td>
			<td>NA</td>
			<td>NA</td>
			<td>60 mM Tris, 100 mM NaCl, 0.5% Triton-X100 (v/v), 1 mM PMSF pH 8.0</td>
		</tr>
		<tr>
			<td>Ni-NTA Resin</td>
			<td></td>
			<td></td>
			<td>6 M urea, 500 mM NaCl, 20 mM Tris, 250 mM imidazole, pH 7.8</td>
		</tr>
		<tr>
			<td>PCR Clean-Up Kit</td>
			<td>Epoch Life Sicences</td>
			<td>2360050</td>
			<td></td>
		</tr>
		<tr>
			<td>Pellet Wash Buffer</td>
			<td>NA</td>
			<td>NA</td>
			<td>60 mM Tris, 100 mM NaCl, pH 8.0</td>
		</tr>
		<tr>
			<td>petDUET-His-SUMO-TEV-H4</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>petDUET-His-TEV-H2A</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>petDUET-His-TEV-H2B</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>petDUET-His-TEV-H3</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>pEVOL-AckRS</td>
			<td>Addgene</td>
			<td>137976</td>
			<td></td>
		</tr>
		<tr>
			<td>pGEM-3z/601</td>
			<td>Addgene</td>
			<td>26656</td>
			<td></td>
		</tr>
		<tr>
			<td>Storage Buffer</td>
			<td>NA</td>
			<td>NA</td>
			<td>20 mM NaCl, 20 mM Tris, 20 mM NaCl, 1 mM EDTA, 20% glycerol, pH 7.8</td>
		</tr>
		<tr>
			<td>Thermocycler</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

site specific lysine acetylation of histones for nucleosome reconstitution using genetic code expansion in escherichia coli

Ацетилированные белки гистона могут быть легко выражены в Escherichia coli кодирования мутанта, Nε-ацетил-лизин (AcK)-специфический Метаносарчина Мази пирролизин тРНК-синтезаз (MmAcKRS1) и его cognate tRNA (tRNAPyl) для сборки восстановленных мононуклеом с сайта конкретных ацетилированных гистонов. MmAcKRS1 и tRNAPyl доставляют AcK на место мутации янтаря в мРНК выбора во время перевода в Escherichia coli. Этот метод широко используется для включения AcK на участках лизина H3. Пирролизил-тРНА синтезаз (PylRS) также может быть легко развивались для включения других неканонических аминокислот (ncAAs) для сайта конкретной модификации белка или функционализации. Здесь мы подробно метод для включения AcK с использованием системы MmAcKRS1 в гистон H3 и интегрировать ацетилированные белки H3 в восстановленных мононуклеозом. Ацетилированные восстановленные мононуклеосомы могут быть использованы в биохимических и связывающих анализах, определении структуры и многое другое. Получение модифицированных мононуклеом имеет решающее значение для разработки экспериментов, связанных с открытием новых взаимодействий и пониманием эпигенетики.

Димеры, тетрамеры и октамеры можно оценить, запуская гель SDS PAGE 12%(рисунок 1 и рисунок 2). Здесь вы можете видеть, что некоторые из ацетилированных теттрамеров имеют более низкую урожайность, чем другие(рисунок 1). На самом д?...

Watch this Scientific Journal Video about Site Specific Lysine Acetylation of Histones for Nucleosome Reconstitution using Genetic Code Expansion in Escherichia coli at JoVE.com

Site Specific Lysine Acetylation of Histones for Nucleosome Reconstitution using Genetic Code Expansion in Escherichia coli

Здесь мы представляем метод выражения ацетилированных белков гистона с помощью расширения генетического кода и собираем восстановленные нуклеосомы в пробирке.

Сайт Конкретные лизин ацетиляции гистонов для нуклеосомы Восстановление с использованием генетического расширения кода в Escherichia coli

Acetylated histone proteins can be easily expressed in Escherichia coli encoding a mutant, N&#949;-acetyl-lysine (AcK)-specific Methanosarcina mazi ...

Obtaining modified mononucleosome is crucial for designing epigenetic experiments. Our protocol provides acetylated reconstituted mononucleosomes that can be used in biochemical and binding assays, structure determination, and more. The primary advantage of our technique is its versatility.This system can be easily modified to incorporate many types of modifications into mononucleosomes. Begin by preparing one liter of autoclaved 2YT media in a culture flask. Inoculate 20 milliliters of the media containing appropriate antibiotics with the co-transformed cell stock that has the tag containing plasmid and pEVOL-AckRS and grow them at 37 degrees Celsius to obtain an OD of 0.6.Add appropriate antibiotics in one liter of autoclaved 2YT media and inoculate it with 20 milliliters of starter culture. Grow the culture at 37 degrees Celsius for two to three hours to obtain an OD of 0.6, 2.8. Add inducing agents and AcK for a final concentration of one millimolar IPTG, 0.2%arabinose, and five millimolar AcK.Grow the culture at 37 degrees Celsius for six to eight hours. Then pellet the cells at 2, 700 x g for 15 minutes and store them overnight at minus 80 degrees Celsius. Dissolve the pellet in 50 milliliters of histone lysis buffer per liter of culture and sonicate the cells at 60%amplitude for three minutes with one second on and one second off intervals.Pellet inclusion bodies at 41, 600 x g for 45 minutes. Discard the supernatant and then resuspend the inclusion bodies in 30 milliliters of histone lysis buffer, then centrifuge again for 30 minutes. Resuspend inclusion bodies again in 30 milliliters of pellet wash buffer and repeat the centrifugation.Next, dissolve inclusion bodies in 25 milliliters of six molar guanidine hydrochloride buffer. Incubate with agitation at 37 degrees for one hour, then centrifuge for 45 minutes. Incubate the supernatant with one milliliter of nickle-NTA resin equilibrated with six molar guanidinium hydrochloride buffer for two hours and proceed with nickle-NTA purification under denatured conditions.Wash the column with three column volumes of wash buffer, then elute acetylated histone protein with 10 milliliters of elution buffer. Dialyze the acetylated histone protein against 5%acetic acid buffer to remove salts for three hours at four degrees Celsius, exchanging the buffer a minimum of six times. Aliquot and lipolyze protein and stored indefinitely at minus 80 degrees.Dissolve aliquots of histone protein pellets H2A, H2B, H3, and H4 so that there's a separate stock of each histone protein in guanidinium hydrochloride buffer for the total volume of 100 microliters. Calculate the concentration of each histone protein by measuring the absorbents at 280 nanometers. If absorbance is greater than one for any protein, dilute with guanidinium hydrochloride buffer to get a more accurate concentration.Combine H2A with H2B and H3 with H4 proteins at a molar ratio of one-to-one and dilute to a total protein concentration of four micrograms per microliter. Dialyze sequentially at four degrees Celsius against two molar TE buffer overnight, one molar TE buffer for two hours and 0.5 molar TE buffer for five hours. When finished, centrifuge at 16, 800 x g at four degrees Celsius to remove the precipitate.Calculate the concentration of histone dimers and tetramers by measuring absorbance at 280 nanometers. Mix the dimers and tetramers at a one-to-one molar ratio and adjust the sodium chloride concentration to 2M. Adjust 601 DNA to two molar TE buffer using 100X TE buffer and solid sodium chloride.Add the solution to the histone octamer at a molar ratio of 0.85, 2.9 to one. Transfer the DNA histone mixture to a dialysis bag and place it at about 200 milliliters of two molar TE buffer with very gentle stirring at four degrees Celsius. Set up peristaltic pump to slowly drip in no-salt TE buffer.When the volume has roughly doubled, pour out half the volume. Do this at least four times, which can take four to eight hours depending on the starting volume. After the salt concentration is reduced to 150 millimolar, dialyze against a 20 millimolar TE buffer overnight.Remove precipitates by centrifugation and measure the concentration of the nucleosomes with a plate reader. Add his-TEV protease and incubate overnight at four degrees Celsius to remove all histidine tags. Then remove the histidine tag impurities from the nucleosome solution with nickel-NTA resin pull down.Incubate at 60 degrees Celsius for one hour to position the nucleosome and homogenize the sample. Store nucleosomes at four degrees Celsius short-term. For long-term storage, dialyze nucleosomes against the storage buffer and store at minus 80 degrees Celsius.Some of the acetylated tetramers had a lower yield than others as assessed by running a 12%SDS-PAGE gel. The closer to the core region, the lower the observed yield. This was most likely due to the acetylation, interfering with the octamer assembly.After assembling octamers with the 601 DNA sequence, the nucleosome can be assessed using a 5%1X TBE-native PAGE gel followed by staining with ethidium bromide. Before thermal positioning, the observed nucleosome bands are very broad and may streak. When performing this protocol, keep the proteins on ice at all times.It is crucial to maintain assembled dimers, tetramers and nucleosome. This procedure has allowed researchers to install the acetyl lysine sites, specifically into mononucleosomes, opening the door to studying post-translational modifications in a chromatin context. It can be easily adapted to install any number of non-canonical amino acids.

Сайт Конкретные лизин ацетиляции гистонов для нуклеосомы Восстановление с использованием генетического расширения кода в Escherichia coli

Abstract