在浦项加速器实验室（韩国）的束线7A处收集X射线衍射数据。这项研究得到了由科学，ICT和未来规划部（2015R1A1A01057574至MH）资助的由韩国国家研究基金会（NRF）管理的基础科学研究计划的支持。

重组蛋白的生产和纯化rBC1531 <ol><li> 制备重组BC1531蛋白（rBC1531）表达质粒 <ol><li>准备蜡样芽孢杆菌的基因组DNA 10 。 </li><li>分别使用正向和反向引物（参见材料列表），通过聚合酶链反应（PCR）从蜡样芽孢杆菌基因组DNA模板扩增bc1531基因，分别产生BamHI和SalI限制酶位点。 </li><li>使用Bam HI和Sal I，如前所述，对PCR产物和修饰的pET49b载体（pET49bm）进行消化 。 </li><li>如前所述，将步骤1.1.3中的消化PCR产物和载体（3:1比例）与T4 DNA连接酶在10μL反应中混合。在18℃孵育30分钟。 </li><li>将3μL连接反应液移取到50μL化学品中将大肠杆菌 DH5α细胞置于管中。通过上下移动轻轻混合，放在冰上30分钟。在42°C下热冲击45秒。加入1mL Luria-Bertani（LB）培养基，并在剧烈振荡（250rpm，37℃，45min）下培养细胞。 </li><li>取100μL来自步骤1.1.6的转化细胞，并用100μg/ mL卡那霉素（Kan）将它们扩散到LB-琼脂平板上。在37°C孵育〜18 h。 </li><li>使用无菌尖端挑取菌落，并在含有100μg/ mL Kan的3mL LB培养基中培养细胞;剧烈振荡（250rpm，37℃，18h）。 </li><li>如上所述，使用碱性SDS裂解法制备质粒DNA。 </li><li>使用DNA测序确认rBC1531表达构建体的核苷酸序列12 。 </li></ol></li><li> rBC1531蛋白的过表达 <ol><li>用rBC1531-exp重新转化大肠杆菌 BL21（DE3）菌株对于过表达，如步骤1.1.5-1.1.6所述。 </li><li>选择菌落并在含有50μg/ mL Kan（LB + Kan）的LB培养基中培养;在37℃下剧烈振荡18小时。 </li><li>在1L LB + Kan培养基中接种10mL的过夜培养物，并在37℃下生长，直到OD 600 （ 600nm的光密度）达到〜0.7。 </li><li>将培养物在冰冷水中浸泡约15分钟，将温度降至18℃，并加入异丙基β-D-1-硫代吡喃半乳糖苷（IPTG）至终浓度为1mM的培养物中用于重组蛋白表达。在18°C下培养另外约16-18小时的培养物。 </li></ol></li><li> 纯化rBC1531 <ol><li>通过离心（5,000×g，4℃，30分钟）收获细胞。仔细丢弃上清液，以免干扰细胞。将细胞重新悬浮在50mL含有10mM咪唑的磷酸盐缓冲盐水（PBS）溶液中。 </li><li>通过在冰上超声处理细胞两次以防止细胞裂解物过度​​加热（时间:2分30秒;周期:5秒;循环:10秒;振幅:38％）。 </li><li>通过离心（约25,000 xg，4℃，30分钟）清除细胞裂解物。 </li><li>混合3 mL镍珠和60 mL含有10 mM咪唑的PBS，并重新流过额外的缓冲液，以在2.5 x 10 cm玻璃色谱柱中沉淀平衡的镍珠。 </li><li>移液以将步骤1.3.3的上清液转移到具有预平衡镍珠的柱上，并在低速（〜20rpm）的旋转轮上在4℃下孵育2小时。 </li><li>允许上清液通过重力排出并用含有10mM咪唑的PBS洗涤柱中的镍珠三次。 </li><li>通过应用含有250mM咪唑的4×5mL PBS洗脱rBC1531蛋白。 </li><li>取15μL步骤1.3.7的洗脱液，并在15％SDS-PAGE上进行洗脱。可视化蛋白质条带凝胶采用考马斯蓝染色10 。 </li></ol></li><li> 通过凝血酶蛋白水解去除rBC1531蛋白的亲和标签 <ol><li>将各部分吸入透析管（分子量切断:3kDa），并将管置于含有4μL凝血酶裂解相容缓冲液（20mM HEPES，pH7.4,150mM NaCl和1.5mMβ-巯基乙醇）的烧杯中，在4℃过夜。 </li><li>移取馏分并将其转移到锥形管中。 </li><li>测量280nm处的吸光度并估计rBC1531蛋白质浓度，如图13所示 。 </li><li>在0.5 mL管中取50μgrBC1531，加入不同量的凝血酶（ 即 2,1,0.6和0.3单位）。将rBC1531和凝血酶反应在20℃孵育〜3小时，以确定切割N端亲和标记所需的凝血酶最少量。 </li><li>运行rBC1531和凝血酶反应15％SDS-PAGE，并确定最终量的凝血酶（ 例如，每50μgrBC1531的0.3单位凝血酶），其完全消化N端亲和标签并产生无标记的rBC1531。 </li><li>在15 mL管中加入10 mg rBC1531蛋白和60单位凝血酶，并在20℃下孵育约3 h，进行凝血酶 - 蛋白水解反应。 </li><li>将凝胶过滤标准品应用于尺寸排阻色谱（SEC）色谱柱，以制备分子量和洗脱体积的标准曲线，如13所述。 </li><li>将来自步骤1.4.6的凝血酶消化的rBC1531蛋白置于离心过滤管（分子量切断:3kDa）中，并在4℃下以3,000g离心以减少至小于5mL的体积。 </li><li>将浓缩的rBC1531蛋白应用于SEC柱，并以每管0.5mL收集60个洗脱级分13 。 </li><li>取15μL每个级分，在15％SDS-PAGE上运行，在使用考马斯染色溶液（0.15％（w / v）考马斯亮蓝，40％（v / v）甲醇和10％（v / v）冰乙酸）的凝胶上。 </li><li>移取含有rBC1531的级分，并将其收集在一个管中。 </li></ol></li></ol> rBC1531的结晶筛选和优化<ol><li> rBC1531蛋白结晶筛选条件 <ol><li>使用离心过滤管将rBC1531从步骤1.4.11浓缩至约18.5mg / mL，如步骤1.4.8所述。 </li><li>将每个结晶筛选溶液（见2.2）的50μL添加到96孔定滴结晶板的孔中。将0.5μL18.5 mg / mL rBC1531蛋白质溶液放在一张坐卧上。将蛋白质溶液与0.5μL井溶液混合，重复整个板的过程。 </li><li>用透明粘合膜覆盖板。将96孔板置于18°C，允许蒸气扩散。 </li><li>使用光学显微镜以20-40倍的放大倍数扫描液滴，每天监测晶体形成2-3周。 </li><li>使用获得的rBC1531蛋白晶体收集X射线衍射数据12 。 </li></ol></li><li> 优化rBC1531结晶条件 <ol><li>准备500μL所选择的初始结晶条件溶液（ 例如， 0.1M二甲胂酸钠，pH 6.5和1.0M柠檬酸钠），并填充24孔结晶板用于坐下水滴。 </li><li>将0.5μL的18.5mg / mL rBC1531蛋白溶液加入到坐式床上，与0.5μL的溶液混合，并用透明的粘合剂膜立即盖住板。将板放在18°C。 </li><li>在光学显微镜下观察晶体生长一周。 </li><li>通过改变pH来优化各种结晶条件（ 即，pH5.5-6.8）和柠檬酸钠的盐浓度（ 即 0.9-1.2M），以获得单晶。监测晶体生长约1周，并收集X射线衍射数据12 。 </li></ol></li></ol> 3.RBC1531的核苷三磷酸酶（NTPase）活性的表征<ol><li> 无机磷酸盐依赖性比色研究的验证 <ol><li>制备焦磷酸钠（100mM），并在200μL反应缓冲液（20mM HEPES，pH 7.4;150μl）中稀释至0.1,0.25,0.5,1.0,5.0,10,50,100和250μM的终浓度mM NaCl;和2mM MgCl 2 ）复制96孔板。使用第一块板作为对照（不含焦磷酸酶）。确保第二块板含有焦磷酸酶作为工作板，如步骤3.1.2中所述。 </li><li>加入0.01单位酿酒酵母无机p将γ-磷酸酶（1μL）加入到工作板的孔中，并在20℃下孵育30-60分钟。 </li><li>通过以7:3的比例混合钼酸铵（0.86％v / v）和抗坏血酸（14％v / v）溶液来制备钼酸盐溶液。在工作井和对照井中加入16μL钼酸溶液，并等待15 min。 </li><li>读取690nm（OD 690nm ）的光密度。 </li></ol></li><li> 比色NTP酶测定 <ol><li>准备100微克核苷三磷酸（NTP）底物，并稀释至所需浓度（ 如 0,0.2,4.4,11,22,44,88或132μM），在180μL测定缓冲液（20mM HEPES， pH 7.4; 150mM NaCl;和2mM MgCl 2 ）。 </li><li>在96孔板中加入20μgrBC1531（1.5mM）和步骤3.2.1中的NTP底物，每个反应最多200μL，上下移液以混合。用盖盖住板，并将其置于37℃的培养箱中30分钟进行底物水解反应。 </li><li>将板转移到70℃的水浴中，静置15分钟以停止rBC1531介导的催化反应。 </li><li>向反应板的每个孔中加入0.01单位的酿酒酵母无机焦磷酸酶（1μL）（步骤3.2.3），并在20℃下孵育30分钟。向反应板中加入16μL钼酸溶液以显色（〜15分钟）。读取OD 690nm 。 </li><li>使用Michaelis-Menten方程进行分析，如第12节所述。 </li></ol></li></ol>

<ol>
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作者没有什么可以披露的。

Studies of true pathogens are limited due to safety restrictions and regulations. Alternatively, pathogens can be studied using evolutionarily related non-pathogens or less pathogenic species. The ba1554 gene is considered a critical gene in B. anthracis. Fortunately, an identical gene is present in nonclinical B. cereus. Thus, without serious safety concerns, recombinant BC1531 protein can be used for biophysical and enzymatic characterization. Here, we described detailed procedures, moving from a gene to a purified recombinant protein. Using recombinant protein, biophysical characterization analyses were conducted to reveal that tetrameric rBC1531 catalyzes NTP hydrolysis into NMP and PPi. 
SEC was employed as the last step of protein purification to remove any protein contaminants derived from the cell lysates that have different sizes than rBC1531. A SEC column was also used to determine the oligomerization status of rBC1531, which was a tetramer. The crystal structure of rBC1531 was consistently determined to be tetrameric12. 
Among biophysical techniques, X-ray crystallography is the best technique for generating atomic models of protein. In general, three-dimensional singular crystals with dimensions larger than 0.1 mm are required for X-ray diffraction. To obtain X-ray diffraction-suitable crystals, crystallization conditions were optimized. For rBC1531, two conditions produced crystals in the initial screening, but only one condition was useful for producing diffraction-suitable crystals12. 
The NTP hydrolysis activity of rBC1531 was assessed by monitoring the formation of a blue-colored complex of molybdate and Pi in the rBC1531-mediated enzymatic reaction15. It has the great advantage of non-radioactively detecting NTP hydrolysis, since the molybdate-Pi complex can be optically measured. In the rBC1531 reaction, the blue-colored complex was generated only in the presence of pyrophosphatase, demonstrating that rBC1531 hydrolyzes NTP into NMP and pyrophosphate. Furthermore, because the blue color of the molybdate-Pi complex can be detected with the naked eye, Pi levels in the rBC1531 reaction, as well as in other applications, can be easily assessed in pilot experiments. Moreover, kinetic parameters, such as Km and Vmax can be calculated by quantifying the intensity of the colorimetric reaction using a visible-light spectrophotometer. However, the presences of free phosphate in the reaction buffer or precipitation during the enzymatic reaction would impair the attainment of accurate results from the NTP hydrolysis reactions. Thus, it is desirable to prepare a Pi standard curve using the identical reaction buffer as in the NTP hydrolysis runs and to ensure an increase in optical density only in the presences of Pi. 
Given that the Km value of the rBC1531-NTP was low (Km = 10 &#181;M), the true substrate for rBC1531 may be a specific type of canonical or noncanonical (d)NTP. To test this possibility, the experimental conditions can be modified to use a specific NTP (i.e., ATP, GTP, CTP, or UTP) rather than a mixture of NTPs. By comparing kinetic parameters, substrate specificity can be determined in future applications. Indeed, additional studies indicate that BC1531 prefers ATP12. Despite the technical simplicity of the colorimetric assay using the molybdate reaction, this assay has a drawback in that the color can become randomly intense when the blue-colored molybdate-Pi complex is left at neutral pH for an extended period of time. Therefore, a critical step is to perform measurements within 15-30 min after the molybdate is added to the Pi-containing sample. The experiment is repeated using the same time point to avoid variations in OD measurements. 
In conclusion, various biophysicochemical methods were used to analyze the oligomerization state and enzymatic activity of rBC1531 and to prepare diffraction-quality crystals for structural studies. These methods can be safely and easily applied to other proteins of interest with minimal experimental equipment requirements.

重组蛋白表达被广泛用于克服与天然蛋白质来源有关的问题，如有限的蛋白质量和有害的污染。此外，在致病基因和蛋白质的研究中，可以使用不需要额外的安全预防措施的替代实验室菌株。例如， 蜡状芽孢杆菌是研究炭疽芽孢杆菌的有用模型，由于它们的进化关系密切。 B.炭疽病是一种专门的病原体，在人类和家畜中引起致命的吸入性炭疽病，可能被用作生物武器2 。因此，炭疽杆菌的实验室研究受到美国疾病控制中心的严格监管，要求生物安全3级（BSL-3）的做法，要求实验室区域以负压力隔离。与炭疽杆菌相反 。，蜡状芽孢杆菌被分类为BSL-1代理，因此具有最小的安全性问题。 蜡状芽孢杆菌是一种机会性病原体，在感染后会导致食物中毒，可以在没有医疗帮助的情况下治疗。然而，由于蜡状 芽孢杆菌与炭疽杆菌共享许多关键基因，可以使用蜡样芽孢杆菌 1的相应同系物研究炭疽杆菌蛋白质的功能。 炭疽杆菌的ba1554 - ba1558基因簇与bc1531 - bc1535簇高度保守 的蜡状 芽孢杆菌 ，以及苏云金芽孢杆菌的bt1364-bt1368簇，在基因组织和序列方面。此外，各簇的第一个基因（ ba1554 ， bc1531和bt1364 ）是绝对保守的（ 即 100％核苷酸序列同一性），暗示在芽孢杆菌属中引起基因产物的关键作用。由于其在这些基因簇中的位置， ba1554被误认为是推定的转录调节因子3 。然而， ba1554产品的氨基酸序列分析表明它属于MazG家族，其具有核苷酸的焦磷酸水解酶活性，与转录因子活性无关4,5 。虽然属于MazG家族的蛋白质在总体序列和长度上是不同的，但它们共有一个共同的〜100个残基的MazG结构域，其特征在于EXXE 12-28 EXXD基序（"X"代表任何氨基酸残基，表示X残基数）。 MazG域并不总是直接涉及某种催化活性。来自大肠杆菌 （EcMazG）的MazG成员具有两个MazG结构域，但是C末端MazG结构域具有酶活性6 。此外，MazG酶的底物特异性从非特异性核苷三磷酸（对于EcMazG）到特异性dCTP / dATP（用于整联蛋白相关MazG）和dUTP（对于dUTP酶） 6,7,8,9不同 。因此，BA1554蛋白的生物物理化学分析是确定其NTPase活性和破译其底物特异性所必需的。 在这里，我们提供了一个分步骤方案，大多数没有BSL-3设施的实验室可以跟踪分子水平上表现为蜡状芽孢杆菌b1515基因的蛋白质产物，这是B. anthracis ba1554的直系同源物。简言之，重组BC1531（rBC1531）在大肠杆菌中表达 ，并使用亲和标签纯化。对于X射线晶体学实验，结晶筛选和优化rBC1531蛋白的定位条件。为了评估rBC1531的酶活性，以比色法监测NTPase活性，以避免常规使用的放射性标记的核苷酸。最后，对获得的生物物理化学数据的分析使我们能够确定rBC1531的低聚状态和催化参数，以及从rBC1531晶体获得X射线衍射数据。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Bacillus cereus ATCC14579</td>
			<td>Korean Collection for Tissue Culture</td>
			<td>3624</td>
			<td></td>
		</tr>
		<tr>
			<td>Genomic DNA prep kit</td>
			<td>GeneAll</td>
			<td>106-101</td>
			<td>Genomic DNA preparation</td>
		</tr>
		<tr>
			<td>Forward primer</td>
			<td>Macrogen</td>
			<td>GAAGGATCCGGAAGCAAAAA 
			CGATGAAAGATATGCA</td>
			<td>BamHI site is underlined</td>
		</tr>
		<tr>
			<td>Reverse primer</td>
			<td>Macrogen</td>
			<td>CTTGTCGACTTATTCTTTCTCT 
			CCCTCATCAATACGTG</td>
			<td>SalI site is underlined</td>
		</tr>
		<tr>
			<td>nPfu-Forte</td>
			<td>Enzynomics</td>
			<td>P410</td>
			<td>PCR polymerase</td>
		</tr>
		<tr>
			<td>BamHI</td>
			<td>Enzynomics</td>
			<td>R003S</td>
			<td></td>
		</tr>
		<tr>
			<td>SalI</td>
			<td>Enzynomics</td>
			<td>R009S</td>
			<td></td>
		</tr>
		<tr>
			<td>pET49b expression vector</td>
			<td>Novagen</td>
			<td>71463</td>
			<td>Expression vector was modified to add affinity tags and BamHI/SalI sites10</td>
		</tr>
		<tr>
			<td>T4 DNA ligase</td>
			<td>Enzynomics</td>
			<td>M001S</td>
			<td></td>
		</tr>
		<tr>
			<td>Dialysis memebrane</td>
			<td>Thermofisher</td>
			<td>18265017</td>
			<td></td>
		</tr>
		<tr>
			<td>Dry block heater</td>
			<td>JSR</td>
			<td>JSBL-02T</td>
			<td></td>
		</tr>
		<tr>
			<td>LB broth (Luria-Bertani)</td>
			<td>LPS solution</td>
			<td>LB-05</td>
			<td></td>
		</tr>
		<tr>
			<td>LB Agar, Miller (Luria-Bertani)</td>
			<td>Becton, Dickinson and Company</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Kanamycin</td>
			<td>LPS solution</td>
			<td>KAN025</td>
			<td></td>
		</tr>
		<tr>
			<td>Mini-prep kit</td>
			<td>Favorgen</td>
			<td>FAPDE300</td>
			<td>Plasmid DNA preparation</td>
		</tr>
		<tr>
			<td>BL21(DE3) Competent cell</td>
			<td>Novagen</td>
			<td>69450-3CN</td>
			<td></td>
		</tr>
		<tr>
			<td>Isopropyl &#946;-D-1-thiogalactopyranoside (IPTG)</td>
			<td>Fisher BioReagents</td>
			<td>50-213-378</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium chloride</td>
			<td>Daejung</td>
			<td>7548-4100</td>
			<td>PBS material</td>
		</tr>
		<tr>
			<td>Potassium chloride</td>
			<td>Daejung</td>
			<td>6566-4405</td>
			<td>PBS material</td>
		</tr>
		<tr>
			<td>Potassium phosphate</td>
			<td>Bio Basic Inc</td>
			<td>PB0445</td>
			<td>PBS material</td>
		</tr>
		<tr>
			<td>Sodium phosphate</td>
			<td>Bio Basic Inc</td>
			<td>S0404</td>
			<td>PBS material</td>
		</tr>
		<tr>
			<td>Imidazole</td>
			<td>Bio Basic Inc</td>
			<td>IB0277</td>
			<td></td>
		</tr>
		<tr>
			<td>Sonicator</td>
			<td>Sonics</td>
			<td>VCX 130</td>
			<td></td>
		</tr>
		<tr>
			<td>Ni-NTA agaros</td>
			<td>Qiagen</td>
			<td>30210</td>
			<td>Nickel bead</td>
		</tr>
		<tr>
			<td>Rotator</td>
			<td>Finepcr</td>
			<td>AG</td>
			<td></td>
		</tr>
		<tr>
			<td>Precision Plus Protein Dual Color Standards</td>
			<td>Bio-rad</td>
			<td>1610374</td>
			<td>SDS-PAGE protein standard</td>
		</tr>
		<tr>
			<td>Coonmassie Brilliant Blue R-250</td>
			<td>Fisher BioReagents</td>
			<td>BP101-25</td>
			<td>Coomassie staining solution</td>
		</tr>
		<tr>
			<td>Methyl alcohol</td>
			<td>Samchun chemical</td>
			<td>M0585</td>
			<td>Coomassie staining solution</td>
		</tr>
		<tr>
			<td>Acetic acid</td>
			<td>Daejung</td>
			<td>1002-4105</td>
			<td>Coomassie staining solution</td>
		</tr>
		<tr>
			<td>Dialysis memebrane</td>
			<td>Thermofisher</td>
			<td>68035</td>
			<td></td>
		</tr>
		<tr>
			<td>2-Mercaptoethanol</td>
			<td>Sigma-aldrich</td>
			<td>M6250</td>
			<td></td>
		</tr>
		<tr>
			<td>Thrombin</td>
			<td>Merckmillipore</td>
			<td>605157-1KUCN</td>
			<td>Prepare aliquots of 20 &#956;l (1 Unit per &#956;l) and store at 70 &#176;C and thaw before use</td>
		</tr>
		<tr>
			<td>Superdex 200 16/600</td>
			<td>General Electric</td>
			<td>28989335</td>
			<td>Size exclusion chromatography</td>
		</tr>
		<tr>
			<td>Gel filtration standard</td>
			<td>Bio-rad</td>
			<td>151-1901</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifugal filter</td>
			<td>Amicon</td>
			<td>UFC800396</td>
			<td></td>
		</tr>
		<tr>
			<td>Crystralization plates</td>
			<td>Hampton research</td>
			<td>HR3-083</td>
			<td>96-well sitting drop plate</td>
		</tr>
		<tr>
			<td>The JCSG core suite I-IV</td>
			<td>Qiagen</td>
			<td>130924-130927</td>
			<td>Crystallization secreening solution</td>
		</tr>
		<tr>
			<td>Microscope</td>
			<td>Nikon</td>
			<td>SMZ745T</td>
			<td></td>
		</tr>
		<tr>
			<td>Cryschem plates</td>
			<td>Hampton research</td>
			<td>HR3-158</td>
			<td>24-well sitting drop plate</td>
		</tr>
		<tr>
			<td>Inorganic pyrophosphatase</td>
			<td>Sigma</td>
			<td>10108987001</td>
			<td></td>
		</tr>
		<tr>
			<td>Ammonium molybdate solution</td>
			<td>Sigma</td>
			<td>13106-76-8</td>
			<td></td>
		</tr>
		<tr>
			<td>L-ascorbic acid</td>
			<td>Sigma</td>
			<td>50-81-7</td>
			<td></td>
		</tr>
		<tr>
			<td>NTP substrate</td>
			<td>Startagene</td>
			<td>200415-51</td>
			<td></td>
		</tr>
		<tr>
			<td>Spectrophotometer</td>
			<td>Biotek</td>
			<td>SynergyH1</td>
			<td>microplate reader</td>
		</tr>
		<tr>
			<td>GraphPad Prism 5</td>
			<td>GraphPad</td>
			<td>Prism 5 for Window</td>
			<td></td>
		</tr>
	</tbody>
</table>

recombinant protein expression, crystallization, and biophysical studies of a bacillus-conserved nucleotide pyrophosphorylase, bcmazg

为了克服从真菌病​​原体研究基因和蛋白质时的安全限制和规定，可以研究其同源物。 炭疽芽孢杆菌是引起致命性吸入性炭疽的专性病原体。 蜡状芽孢杆菌被认为是研究炭疽杆菌由于其进化关系密切的有用模型。 炭疽杆菌的基因簇ba1554 -ba1558与枯草芽孢杆菌中的bc1531-bc1535簇以及苏云金芽孢杆菌中的bt1364-bt1368簇具有高度保守性，表明了相关基因在芽孢杆菌属中的关键作用。该手稿描述了使用其在蜡样 芽孢杆菌bc1531中其直向同源物的重组蛋白质来制备和表征来自炭疽杆菌的基因簇的第一基因（ ba1554 ）的蛋白质产物的方法。

本研究中所关注的蛋白质的表征开始于制备足量的重组乙型肝炎杆菌bc1531 （rBC1531）蛋白，优选多于几毫克。通过使用蜡状芽孢杆菌的基因组DNA作为模板，通过PCR制备编码BC1531蛋白的DNA片段，因为它含有与ba1554相同的直系同源基因。 rBC1531在大肠杆菌细胞中被过表达为可溶性蛋白。 rBC1531蛋白在其N末端12处用6x-组氨酸标签和凝血酶切割位点表达。作为纯化的第一步，通过镍亲和层析纯化rBC1531（ 图1A ）。接下来，进行凝血酶消化试验以确定从rBC1531完全消化亲和标签所需的凝血酶的最低量（ 图1A ）。在我们手中，0.3单位的凝血酶是suf无法从rBC1531中完全删除亲和性标签。因此，当结垢时，用60单位凝血酶处理10mg rBC1531以进行标签去除。凝血酶消化后，将无标记的rBC1531应用于SEC柱以除去任何可溶性聚集体和较高或较低分子量的污染物。通过SDS-PAGE分析rBC1531级分，结果表明rBC1531纯度为〜99％（ 图1A ）。总体而言，1升培养物产生约8mg的rBC1531蛋白。 为了估计rBC1531的寡聚状态，进行SEC。将样品分子量对数值与其相应洗脱体积相关联的标准线性曲线使用蛋白质标准物的峰进行绘制（ 图1B ）。 rBC1531以约84mL的洗脱体积洗脱，其表观分子量估计为〜55kDa。鉴于计算的分子我们rBC1531单体的亮度约为13kDa，这些结果表明rBC1531组装为四聚体。 纯化的rBC1531筛选结晶使用约400条件的坐式蒸气扩散法。在两个条件下，rBC1531晶体出现在〜2天:条件-A，0.1M柠檬酸钠（pH 5.5）和20％PEG 3000（ 图2A ）和条件-B，0.1M二甲胂酸钠（pH6.5）和1.0M钠柠檬酸盐（ 图2B ）。通过从条件-A改变pH（0.1M柠檬酸钠，pH 5.0-6.0）和PEG 3000（18-21％PEG 3000）的浓度并通过改变pH（0.1M二聚氰酸钠，pH值）来优化结晶条件5.5-6.8）和盐浓度（0.9-1.2M柠檬酸钠）。仅对条件B获得衍射合适的晶体，而条件-A的晶体往往是生长而不是单个的。条件B晶体衍射X射线分辨率为2.74Å（ 图2C ），并用于rBC1531结构测定。 BA1554蛋白序列中保守的MazG结构域的观察表明存在能够水解NTPs的NTPase活性。 NTP水解通常产生核苷二磷酸（NDP）和无机磷酸（Pi）或核苷单磷酸（NMP）和焦磷酸（PPi）。 PPi需要额外的焦磷酸酶活性来产生Pi。可以使用钼酸盐直接测量Pi的水平，钼酸盐与Pi（钼酸盐-Ib）形成蓝色络合物，可以使用波长690nm（OD 690nm ）的光密度监测 （ 图3A ）。在我们的研究中，在通过rBC1531进行NTP水解后，在加入钼酸盐后没有产生可见的蓝色（ 图3B ）。然而，当将焦磷酸酶加入到rBC1531介导的NTP水解r中时色素变为深蓝色（ 图3B ），表明rBC1531具有NTP焦磷酸水解酶的活性。使用非线性回归方法分析OD 690nm和NTP浓度的图，以确定rBC1531酶的动力学参数（Vmax为0.75和K m为10μM）（ 图3C ）。 <img alt="图1" src="/files/ftp_upload/55576/55576fig1.jpg" /> 图1:SDS-PAGE和大小排阻层析（SEC）分析。 （ A ）rBC1531的SDS-PAGE分析。 （左）与蛋白标准品（泳道1;标记为kDa）一起分析来自镍亲和层析（泳道2，Ni）和SEC（泳道3）的rBC1531洗脱峰。 （右）分析用于去除亲和标签的rBC1531的凝血酶消化。在不同反应中加入到50μgrBC1531中的凝血酶量为ind凝结在凝胶上方。 （ B ）rBC1531的大小排阻色谱（SEC）分析和蛋白质标准。 （左）rBC1531（蓝色）和标准（红色）的洗脱曲线。标准蛋白的分子量显示在每个峰上方，以kDa表示。垂直（y）和水平（x）轴分别显示毫摩尔吸收单位（mAU在280nm）和保留体积（mL）。 （右）使用线性图估计rBC1531的表观分子大小，分子量，对数标度和蛋白质标准品的洗脱体积（R 2 = 0.9934）。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55576/55576fig1large.jpg" target="_blank">请点击此处查看此图的较大版本。</a> <img alt="图2" src="/files/ftp_upload/55576/55576fig2.jpg" /> 图2:结晶和X射线衍射。 （ A ）条件下的rBC1531晶体（ B ）在条件B，0.1M二甲胂酸钠（pH6.5）和1.0M柠檬酸钠中的rBC1531晶体。显示初始（左）和优化（中，右）晶体。将优化的rBC1531晶体（右，〜0.35mm×0.35mm×0.20mm）用于X射线衍射。刻度棒=100μm。 （ C ）在PAL光束线BL-7A 12处获得的rBC1531晶体的X射线衍射图像。箭头表示高分辨率点。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55576/55576fig2large.jpg" target="_blank">请点击此处查看此图的较大版本。</a> <img alt="图3" src="/files/ftp_upload/55576/55576fig3.jpg" /> 图3:NTPase活动。 （ A ）钼-pi络合物的形成取决于Pi浓度。 690nm处的光密度为di与Pi水平的增加直接相关。 Y轴和X轴分别表示Pi的OD 690nm和浓度（μM）。 （ B ）添加焦磷酸酶后颜色发生变化。在第一排孔中，没有加入焦磷酸酶，不形成蓝色的Pi-复合物。然而，第二排由其中rBC1531-NTP反应用焦磷酸酶处理并开发出蓝色的孔组成。 OD 690nm值随着NTP浓度的增加而增加。 （ C ）rBC1531-NTP反应的Michaelis-Menten图。 Y轴和X轴分别表示NTP底物的OD 690nm和浓度（0-120μM）。误差棒代表三个单独实验的标准偏差。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55576/55576fig3large.jpg" target="_blank">请点击此处查看此图的较大版本。</a>

Watch this Scientific Journal Video about Recombinant Protein Expression, Crystallization, and Biophysical Studies of a Bacillus-conserved Nucleotide Pyrophosphorylase, BcMazG at JoVE.com

Recombinant Protein Expression, Crystallization, and Biophysical Studies of a Bacillus-conserved Nucleotide Pyrophosphorylase, BcMazG

炭疽芽孢杆菌是致命性吸入性炭疽的专性病原体，因此对其基因和蛋白质的研究受到严格管制。另一种方法是研究直系同源基因。我们描述了蜡状芽孢杆菌Bc1531中的炭疽杆菌直系同源物的生物物理化学研究，需要最少的实验设备和缺乏严重的安全性问题。

重组蛋白表达，结晶和生物物理研究<em&gt;芽孢杆菌</em&gt; - 保守的核苷酸焦磷酸化酶，BcMazG

To overcome safety restrictions and regulations when studying genes and proteins from true pathogens, their homologues can be studied. Bacillus anthracis ...

recombinant-protein-expression-crystallization-biophysical-studies

Nanyang Technological University

Research

JoVE Journal

Biochemistry

Recombinant Protein Expression, Crystallization, and Biophysical Studies of a Bacillus-conserved Nucleotide Pyrophosphorylase, BcMazG

7.4K 次观看.  Yonsei University.  炭疽芽孢杆菌是致命性吸入性炭疽的专性病原体，因此对其基因和蛋白质的研究受到严格管制。另一种方法是研究直系同源基因。我们描述了蜡状芽孢杆菌Bc1531中的炭疽杆菌直系同源物的生物物理化学研究，需要最少的实验设备和缺乏严重的安全性问题。 

视频: Recombinant Protein Expression, Crystallization, and Biophysical Studies of a Bacillus-conserved Nucleotide Pyrophosphorylase, BcMazG

Thermostabilization, Expression, Purification, and Crystallization of the Human Serotonin Transporter Bound to S-citalopram

The serotonin transporter is a sodium and chloride-coupled transporter that &#34;pumps&#34; extracellular serotonin into cells. S-citalopram is a drug used to treat depression and anxiety by binding to the serotonin transporter with high-affinity, blocking serotonin reuptake. Here we report an efficient procedure and a set of tools to stabilize, express, purify, and crystallize serotonin transporter-antibody complexes bound to S-citalopram and other antidepressants. Mutations which stabilize the serotonin transporter were identified using an S-citalopram binding assay. Serotonin transporter expressed in baculovirus-transduced HEK293S GnTI- cells, was reconstituted into proteoliposomes and used to raise high-affinity antibodies. We have developed a strategy to discover antibodies that are useful for structural studies. A straightforward approach for the expression of antibody fragments in Sf9 cells has also been established. Transporter-antibody complexes purified using this procedure are well-behaved and readily crystallize, producing complexes with S-citalopram that diffract X-rays to 3-4 &#197; resolution. The strategies developed here can be utilized to determine the structure of other challenging membrane proteins.

Thermostabilization, Expression, Purification, and Crystallization of the Human Serotonin Transporter Bound to S-citalopram

This manuscript describes how to screen for thermostabilizing mutations, purify the human serotonin transporter, generate high affinity antibodies, and crystallize the serotonin transporter-antibody complex bound to the antidepressant drug S-citalopram. This protocol can be adapted to the study of other challenging membrane transporters, receptors, and channels.

热稳定，表达，纯化，和人类血清素转运蛋白结晶绑定到<em&gt;取值</em&gt; -citalopram

The serotonin transporter is a sodium and chloride-coupled transporter that &#34;pumps&#34; extracellular serotonin into cells. S-citalopram is a drug ...

科研

生物化学

Production, Crystallization and Structure Determination of C. difficile PPEP-1 via Microseeding and Zinc-SAD

New therapies are needed to treat Clostridium difficile infections that are a major threat to human health. The C. difficile metalloprotease PPEP-1 is a target for future development of inhibitors to decrease the virulence of the pathogen. To perform biophysical and structural characterization as well as inhibitor screening, large amounts of pure and active protein will be needed. We have developed a protocol for efficient production and purification of PPEP-1 by the use of E. coli as the expression host yielding sufficient amounts and purity of protein for crystallization and structure determination. Additionally, using microseeding, highly intergrown crystals of PPEP-1 can be grown to well-ordered crystals suitable for X-ray diffraction analysis. The methods could also be used to produce other recombinant proteins and to study the structures of other proteins producing intergrown crystals.

Production, Crystallization and Structure Determination of C. difficile PPEP-1 via Microseeding and Zinc-SAD

Proline-proline endopeptidase-1 (PPEP-1) is a secreted metalloprotease and promising drug-target from the human pathogen Clostridium difficile. Here we describe all methods necessary for the production and structure determination of this protein.

生产，结晶和结构测定<em&gt;℃。难辨</em&gt;通过Microseeding和锌SAD PPEP-1

New therapies are needed to treat Clostridium difficile infections that are a major threat to human health. The C. difficile metalloprotease PPEP-1 is a ...

A Modified Yeast-one Hybrid System for Heteromeric Protein Complex-DNA Interaction Studies

Over the years, the yeast one-hybrid assay has proven to be an important technique for the identification and validation of physical interactions between proteins such as transcription factors (TFs) and their DNA target. The method presented here utilizes the underlying concept of the Y1H but is modified further to study and validate protein complexes binding to their target DNA. Hence, it is referred to as the modified yeast one-hybrid (Y1.5H) assay. This assay is cost effective and can be easily performed in a regular laboratory setting. Albeit using a heterologous system, the described method could be a valuable tool to test and validate the heteromeric protein complex binding to their DNA target(s) for functional genomics in any system of study, especially plant genomics.

The modified yeast one-hybrid assay described here is an extension of the classical yeast one-hybrid (Y1H) assay to study and validate the heteromeric protein complex-DNA interaction in a heterologous system for any functional genomics study.

用于异构蛋白复合物 -  DNA相互作用研究的修饰的酵母一杂交系统

Over the years, the yeast one-hybrid assay has proven to be an important technique for the identification and validation of physical interactions between ...

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

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 is a novel transcription activator found specifically in the obligate intracellular bacterial pathogen Chlamydia. Protein pulldown assays using affinity beads have revealed that GrgA binds two &#963; factors, namely &#963;66 and &#963;28, which recognize different sets of promoters for genes whose products are differentially required at developmental stages. We have used BLI to confirm and further characterize the interactions. BLI demonstrates several advantages over pulldown: 1) It reveals real-time association and dissociation between binding partners, 2) It generates quantitative kinetic parameters, and 3) It can detect bindings that pulldown assays often fail to detect. These characteristics have enabled us to deduce the physiological roles of GrgA in gene expression regulation in Chlamydia, and possible detailed interaction mechanisms. We envision that this relatively affordable technology can be extremely useful for studying transcription and other biological processes.

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

Interactions of transcription factors (TFs) with the RNA polymerase are usually studied using pulldown assays. We apply a Biolayer Interferometry (BLI) technology to characterize the interaction of GrgA with the chlamydial RNA polymerase. Compared to pulldown assays, BLI detects real-time association and dissociation, offers higher sensitivity, and is highly quantitative.

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 ...

Expression, Purification, Crystallization, and Enzyme Assays of Fumarylacetoacetate Hydrolase Domain-Containing Proteins

Fumarylacetoacetate hydrolase (FAH) domain-containing proteins (FAHD) are identified members of the FAH superfamily in eukaryotes. Enzymes of this superfamily generally display multi-functionality, involving mainly hydrolase and decarboxylase mechanisms. This article presents a series of consecutive methods for the expression and purification of FAHD proteins, mainly FAHD protein 1 (FAHD1) orthologues among species (human, mouse, nematodes, plants, etc.). Covered methods are protein expression in E. coli, affinity chromatography, ion exchange chromatography, preparative and analytical gel filtration, crystallization, X-ray diffraction, and photometric assays. Concentrated protein of high levels of purity (&#62;98%) may be employed for crystallization or antibody production. Proteins of similar or lower quality may be employed in enzyme assays or used as antigens in detection systems (Western-Blot, ELISA). In the discussion of this work, the identified enzymatic mechanisms of FAHD1 are outlined to describe its hydrolase and decarboxylase bi-functionality in more detail.

Expression and purification of fumarylacetoacetate hydrolase domain-containing proteins is described with examples (expression in E. coli, FPLC). Purified proteins are used for crystallization and antibody production and employed for enzyme assays. Selected photometric assays are presented to display the multi-functionality of FAHD1 as oxaloacetate decarboxylase and acylpyruvate hydrolase.

富玛莱酸氢酶含含蛋白质的表达、纯化、结晶和酶测定

Fumarylacetoacetate hydrolase (FAH) domain-containing proteins (FAHD) are identified members of the FAH superfamily in eukaryotes. Enzymes of this ...

Fully Processed Recombinant KRAS4b: Isolating and Characterizing the Farnesylated and Methylated Protein

Protein prenylation is a key modification that is responsible for targeting proteins to intracellular membranes. KRAS4b, which is mutated in 22% of human cancers, is processed by farnesylation and carboxymethylation due to the presence of a &#39;CAAX&#39; box motif at the C-terminus. An engineered baculovirus system was used to express farnesylated and carboxymethylated KRAS4b in insect cells and has been described previously. Here, we describe the detailed, practical purification and biochemical characterization of the protein. Specifically, affinity and ion exchange chromatography were used to purify the protein to homogeneity. Intact and native mass spectrometry was used to validate the correct modification of KRAS4b and to verify nucleotide binding. Finally, membrane association of farnesylated and carboxymethylated KRAS4b to liposomes was measured using surface plasmon resonance spectroscopy.

Prenylation is an important modification on peripheral membrane binding proteins. Insect cells can be manipulated to produce farnesylated and carboxymethylated KRAS4b in quantities that enable biophysical measurements of protein-protein and protein-lipid interactions

完全加工重组KRAS4b:分离和描述脱脂和甲基化蛋白

Protein prenylation is a key modification that is responsible for targeting proteins to intracellular membranes. KRAS4b, which is mutated in 22% of human ...

Strategic Screening and Characterization of the Visual GPCR-mini-G Protein Signaling Complex for Successful Crystallization

The key to determining crystal structures of membrane protein complexes is the quality of the sample prior to crystallization. In particular, the choice of detergent is critical, because it affects both the stability and monodispersity of the complex. We recently determined the crystal structure of an active state of bovine rhodopsin coupled to an engineered G protein, mini-Go, at 3.1 &#197; resolution. Here, we detail the procedure for optimizing the preparation of the rhodopsin&#8211;mini-Go complex. Dark-state rhodopsin was prepared in classical and neopentyl glycol (NPG) detergents, followed by complex formation with mini-Go under light exposure. The stability of the rhodopsin was assessed by ultraviolet-visible (UV-VIS) spectroscopy, which monitors the reconstitution into rhodopsin of the light-sensitive ligand, 9-cis retinal. Automated size-exclusion chromatography (SEC) was used to characterize the monodispersity of rhodopsin and the rhodopsin&#8211;mini-Go complex. SDS-polyacrylamide electrophoresis (SDS-PAGE) confirmed the formation of the complex by identifying a 1:1 molar ratio between rhodopsin and mini-Go after staining the gel with Coomassie blue. After cross-validating all this analytical data, we eliminated unsuitable detergents and continued with the best candidate detergent for large-scale preparation and crystallization. An additional problem arose from the heterogeneity of N-glycosylation. Heterologously-expressed rhodopsin was observed on SDS-PAGE to have two different N-glycosylated populations, which would probably have hindered crystallogenesis. Therefore, different deglycosylation enzymes were tested, and endoglycosidase F1 (EndoF1) produced rhodopsin with a single species of N-glycosylation. With this strategic pipeline for characterizing protein quality, preparation of the rhodopsin&#8211;mini-Go complex was optimized to deliver the crystal structure. This was only the third crystal structure of a GPCR&#8211;G protein signaling complex. This approach can also be generalized for other membrane proteins and their complexes to facilitate sample preparation and structure determination.

This report describes screening of different detergents for preparing the visual GPCR, rhodopsin, and its complex with mini-Go. Biochemical methods characterizing the quality of the complex at different stages during purification are demonstrated. This protocol can be generalized to other membrane protein complexes for their future structural studies.

视觉GPCR-迷你G蛋白信号复合物的战略筛选与表征，成功结晶

The key to determining crystal structures of membrane protein complexes is the quality of the sample prior to crystallization. In particular, the choice ...

PCR Mutagenesis, Cloning, Expression, Fast Protein Purification Protocols and Crystallization of the Wild Type and Mutant Forms of Tryptophan Synthase

Structural studies with tryptophan synthase (TS) bienzyme complex (&#945;2&#946;2 TS) from Salmonella typhimurium have been performed to better understand its catalytic mechanism, allosteric behavior, and details of the enzymatic transformation of substrate to product in PLP-dependent enzymes. In this work, a novel expression system to produce the isolated &#945;- and isolated &#946;-subunit allowed the purification of high amounts of pure subunits and &#945;2&#946;2 StTS complex from the isolated subunits within 2 days. Purification was carried out by affinity chromatography followed by cleavage of the affinity tag, ammonium sulfate precipitation, and size exclusion chromatography (SEC). To better understand the role of key residues at the enzyme &#946;-site, site-direct mutagenesis was performed in prior structural studies. Another protocol was created to purify the wild type and mutant &#945;2&#946;2 StTS complexes. A simple, fast and efficient protocol using ammonium sulfate fractionation and SEC allowed purification of &#945;2&#946;2 StTS complex in a single day. Both purification protocols described in this work have considerable advantages when compared with previous protocols to purify the same complex using PEG 8000 and spermine to crystalize the &#945;2&#946;2 StTS complex along the purification protocol. Crystallization of wild type and some mutant forms occurs under slightly different conditions, impairing the purification of some mutants using PEG 8000 and spermine. To prepare crystals suitable for x-ray crystallographic studies several efforts were made to optimize crystallization, crystal quality and cryoprotection. The methods presented here should be generally applicable for purification of tryptophan synthase subunits and wild type and mutant &#945;2&#946;2 StTS complexes.

This article presents a series of consecutive methods for the expression and purification of Salmonella typhimurium tryptophan synthase comp this protocol a rapid system to purify the protein complex in a day. Covered methods are site-directed mutagenesis, protein expression in Escherichia coli, affinity chromatography, gel filtration chromatography, and crystallization.

PCR 突变体、克隆、表达、快速蛋白质纯化协议和色氨酸合成酶野生类型和突变形式的结晶

Structural studies with tryptophan synthase (TS) bienzyme complex (&#945;2&#946;2 TS) from Salmonella typhimurium have been performed to better understand ...

Production of Recombinant PRMT Proteins using the Baculovirus Expression Vector System

Protein arginine methyltransferases (PRMTs) methylate arginine residues on a wide variety of proteins that play roles in numerous cellular processes. PRMTs can either mono- or dimethylate arginine guanidino groups symmetrically or asymmetrically. The enzymology of these proteins is a complex and intensely investigated area that requires milligram quantities of high-quality recombinant protein. The baculovirus expression vector system (BEVS) employing Autographa californica multiple nucleopolyhedrovirus (AcMNPV) and Spodoptera frugiperda 9 (Sf9) insect cells has been used for expression screening and production of many PRMTs, including PRMT 1, 2, and 4 through 9. To simultaneously screen for the expression of multiple constructs of these proteins, including domains and truncated fragments as well as the full-length proteins, we have applied scalable methods utilizing adjustable and programmable multichannel pipettes, combined with 24- and 96-well plates and blocks. Overall, these method adjustments enabled a large-scale generation of bacmid DNA, recombinant viruses, and protein expression screening. Using culture vessels with a high-fill volume of Sf9 cell suspension helped to overcome space limitations in the production pipeline for single batch large-scale protein production. Here, we describe detailed protocols for the efficient and cost-effective expression of functional PRMTs for biochemical, biophysical, and structural studies.

The baculovirus expression vector system (BEVS) is a robust platform for expression screening and production of protein arginine methyltransferases (PRMTs) to be used for biochemical, biophysical, and structural studies. Milligram quantities of material can be produced for the majority of PRMTs and other proteins of interest requiring a eukaryotic expression platform.

使用巴库洛病毒表达载体系统生产重组PRMT蛋白

Protein arginine methyltransferases (PRMTs) methylate arginine residues on a wide variety of proteins that play roles in numerous cellular processes. ...

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

With recent advances in mass spectrometry-based proteomics technologies, deep profiling of hundreds of proteomes has become increasingly feasible. However, deriving biological insights from such valuable datasets is challenging. Here we introduce a systems biology-based software JUMPn, and its associated protocol to organize the proteome into protein co-expression clusters across samples and protein-protein interaction (PPI) networks connected by modules (e.g., protein complexes). Using the R/Shiny platform, the JUMPn software streamlines the analysis of co-expression clustering, pathway enrichment, and PPI module detection, with integrated data visualization and a user-friendly interface. The main steps of the protocol include installation of the JUMPn software, the definition of differentially expressed proteins or the (dys)regulated proteome, determination of meaningful co-expression clusters and PPI modules, and result visualization. While the protocol is demonstrated using an isobaric labeling-based proteome profile, JUMPn is generally applicable to a wide range of quantitative datasets (e.g., label-free proteomics). The JUMPn software and protocol thus provide a powerful tool to facilitate biological interpretation in quantitative proteomics.

We present a systems biology tool JUMPn to perform and visualize network analysis for quantitative proteomics data, with a detailed protocol including data pre-processing, co-expression clustering, pathway enrichment, and protein-protein interaction network analysis.