웨슬리 왕 박사에게 이 프로토콜과 그의 소중한 멘토링에 대한 기초를 마련해 주신 것에 감사드립니다. 이 작품은 부분적으로 건강의 국가 학회에 의해 지원되었다 (보조금 R01GM127575 및 R01GM121584) 및 웰치 재단 (그랜트 A-1715).

1. 플라스미드 건설
<ol><li>어떤 히스톤 단백질이 아세틸화되고 어떤 리신 부위를 결정할지 결정하십시오. 사이트 지시 돌연변이 발생을 사용하여 황색 정지 코돈 (TAG)에 사이트를 돌연변이. 참고: 히스톤 단백질의 발현에 활용된 4개의 이전에 디자인된 플라스미드가 있습니다. 4개의 히스톤 단백질은 모두 N-단말 히스티딘 태그로 pETDuet-1 벡터로 복제하였다. Histone H4에는 또한 SUMO 태그, 약 40의 ?...

<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. 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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>

실험 중에 이 프로토콜을 모든 세부 사항으로 따르는 것이 필수적입니다. 뉴클레오좀은 매우 안정적이지 않으며 많은 시행착오가 이 프로토콜을 결정하는 데 들어갔습니다. 미립자가 조립 공정을 쉽게 방해할 수 있기 때문에 모든 단계(또는 관찰 될 때마다)에서 침전을 제거하는 것이 중요합니다. 항상 얼음에 히스톤 샘플을 유지합니다. 뉴클레오좀이 너무 오래 4°C로 저장되면 자발적으로 분해?...

우리는 PylRS와 tRNAPyl을 사용하여 사이트 특정 아세틸화 히스톤으로 재구성된 단핵구를 합성하고 조립했습니다. PylRS는 포스트 번역 변형 (PTM)을 가진 단백질을 생성하는 유전 코드 확장 도구로 귀중한 입증하고 약 200 다른 ncAA를 통합하기 위해 유전적으로 진화되었습니다. PylRS는 호박색 정지 코돈에 통합되어 번역 중에 다른 아미노산에서 경쟁을 제거합니다. PylRS는 메탄원성 고고학에서 처음 발견되었으며, 이후 화학 생물학에서 새로운 반응성 화학 그룹을 단백질1,2에통합하는 데 활용되었습니다.
MmAcKRS1은 메타노사르키나 미제이 PylRS로부터 진화하고 아세틸화된 단백질3,4,5,6의합성을 위해 실험실에서 자주 사용되었다. MmAcKRS1은 대장균에서 번역하는 동안 선택되는 mRNA에 있는 호박색 돌연변이 부위에서 AcK를 전달한다. 이 기술은 이전에 K4, K9, K14, K18, K23, K27, K36, K56, K64, K79 히스톤 H3 리신 부위에 AcK를 통합하여 1, 2, 6, 7의 아세트 모노뉴클레오솜4,5,6의AcK를 통합하는 데 사용되었습니다. 여기서 우리는 아세틸화 된 히스톤을 발현하고 아세틸화 뉴클레오좀을 재구성하는이 방법을 자세히 설명합니다.

<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

아세틸화 히스톤 단백질은 돌연변이, N ε-아세틸-리신(AcK)-특이적 메타노사르시나 마지 피롤리신 tRNA-synthetase(MmAcKRS1) 및 그 코그네이트 tRNA(tRNAPyl)를결합하여 재구성한 단핵형 특이적 군수로 재구성할 수 있는 에셀리치아 대장균에서 용이하게 발현될 수 있다. MmAcKRS1 및 tRNAPyl은 대장균에서 번역하는 동안 선택되는 mRNA에 있는 황색 돌연변이 부위에서 AcK를 전달한다. 이 기술은 H3 lysine 사이트에 AcK를 통합하는 데 광범위하게 사용되었습니다. Pyrrolysyl-tRNA synthetase (PylRS)는 또한 사이트 특정 단백질 수정 또는 기능화를 위한 다른 비카노마이컬 아미노산(ncAA)을 통합하도록 쉽게 진화될 수 있다. 여기서 우리는 MmAcKRS1 시스템을 사용하여 AcK를 히스톤 H3에 통합하고 아세틸화 된 H3 단백질을 재구성 된 단핵구로 통합하는 방법을 자세히 설명합니다. 아세틸화 재구성 된 단핵구는 생화학 적 및 결합 적 반응, 구조 결정 등에 사용될 수 있습니다. 수정된 단핵구를 얻는 것은 새로운 상호 작용을 발견하고 후성 유전학을 이해하는 관련있는 실험을 디자인하기 위해 중요합니다.

디머, 테트라머 및 옥타머는 12% SDS PAGE젤(그림 1 및 도 2)을실행하여 평가할 수 있다. 여기서 당신은 아세틸화 테트라머중 일부가 다른 테트라머보다 낮은 수율을 가지고 있음을 알 수 있습니다(그림 1). 사실, 코어 영역에 가까울수록 수율이 낮아집니다. 이것은 가장 가능성이 있기 때문에 테트라머의 조립?...

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

여기서 우리는 유전 코드 확장을 사용하여 아세틸화 히스톤 단백질을 발현하고 체외에서 재구성된 뉴클레오좀을 조립하는 방법을 제시한다.

에체리치아 대장균의 유전코드 확장을 이용한 뉴클레오소성 재구성을 위한 히스톤의 특정 리신 아세틸화

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.