Name: recombinant protein expression, crystallization, and biophysical studies of a bacillus-conserved nucleotide pyrophosphorylase, bcmazg
Uploaded: 2017-05-16T13:00:00.000+00:00
Duration: 12 min 23 s
Description: Watch this Scientific Journal Video about Recombinant Protein Expression, Crystallization, and Biophysical Studies of a Bacillus-conserved Nucleotide Pyrophosphorylase, BcMazG at JoVE.com

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>

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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 fr...

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><tbody><tr><td>&lt;em&gt;Bacillus cereus&lt;/em&gt; 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&lt;br/&gt; CGATGAAAGATATGCA</td><td>&lt;em&gt;Bam&lt;/em&gt;HI site is underlined</td></tr><tr><td>Reverse primer</td><td>Macrogen</td><td>CTTGTCGACTTATTCTTTCTCT&lt;br/&gt; CCCTCATCAATACGTG</td><td>&lt;em&gt;Sal&lt;/em&gt;I site is underlined</td></tr><tr><td>nPfu-Forte</td><td>Enzynomics</td><td>P410</td><td>PCR polymerase</td></tr><tr><td>&lt;em&gt;Bam&lt;/em&gt;HI</td><td>Enzynomics</td><td>R003S</td><td></td></tr><tr><td>&lt;em&gt;Sal&lt;/em&gt;I</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 sites&lt;sup&gt;10&lt;/sup&gt;</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 &amp;beta;-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 &amp;mu;L (1 Unit per &amp;mu;L) and store at 70 &amp;deg;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?...

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

Universidad San Sebastian

Research

JoVE Journal

Biochemistry

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

7.5K 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

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 ...

科研

生物化学

Microcrystallography of Protein Crystals and In Cellulo Diffraction

The advent of high-quality microfocus beamlines at many synchrotron facilities has permitted the routine analysis of crystals smaller than 10 &#181;m in their largest dimension, which used to represent a challenge. We present two alternative workflows for the structure determination of protein microcrystals by X-ray crystallography with a particular focus on crystals grown in vivo. The microcrystals are either extracted from cells by sonication and purified by differential centrifugation, or analyzed in cellulo after cell sorting by flow cytometry of crystal-containing cells. Optionally, purified crystals or crystal-containing cells are soaked in heavy atom solutions for experimental phasing. These samples are then prepared for diffraction experiments in a similar way by application onto a micromesh support and flash cooling in liquid nitrogen. We briefly describe and compare serial diffraction experiments of isolated microcrystals and crystal-containing cells using a microfocus synchrotron beamline to produce datasets suitable for phasing, model building and refinement.
These workflows are exemplified with crystals of the Bombyx mori cypovirus 1 (BmCPV1) polyhedrin produced by infection of insect cells with a recombinant baculovirus. In this case study, in cellulo analysis is more efficient than analysis of purified crystals and yields a structure in ~8 days from expression to refinement.

Microcrystallography of Protein Crystals and In Cellulo Diffraction

A protocol is presented for X-ray crystallography using protein microcrystals. Two examples analyzing in vivo-grown microcrystals after purification or in cellulo are compared.

蛋白晶体的微结晶和<em&gt;在Cellulo</em&gt;衍射

The advent of high-quality microfocus beamlines at many synchrotron facilities has permitted the routine analysis of crystals smaller than 10 &#181;m in ...

Expression and Purification of Mammalian Bestrophin Ion Channels

The human genome encodes four bestrophin paralogs, namely BEST1, BEST2, BEST3, and BEST4. BEST1, encoded by the BEST1 gene, is a Ca2+-activated Cl- channel (CaCC) predominantly expressed in retinal pigment epithelium (RPE). The physiological and pathological significance of BEST1 is highlighted by the fact that over 200 distinct mutations in the BEST1 gene have been genetically linked to a spectrum of at least five retinal degenerative disorders, such as Best vitelliform macular dystrophy (Best disease). Therefore, understanding the biophysics of bestrophin channels at the single-molecule level holds tremendous significance. However, obtaining purified mammalian ion channels is often a challenging task. Here, we report a protocol for the expression of mammalian bestrophin proteins with the BacMam baculovirus gene transfer system and their purification by affinity and size-exclusion chromatography. The purified proteins have the potential to be utilized in subsequent functional and structural analyses, such as electrophysiological recording in lipid bilayers and crystallography. Importantly, this pipeline can be adapted to study the functions and structures of other ion channels.

The purification of ion channels is often challenging, but once achieved, it can potentially allow in vitro investigations of the functions and structures of the channels. Here, we describe the stepwise procedures for the expression and purification of mammalian bestrophin proteins, a family of Ca2+-activated Cl- channels.

哺乳动物 Bestrophin 离子通道的表达与纯化

The human genome encodes four bestrophin paralogs, namely BEST1, BEST2, BEST3, and BEST4. BEST1, encoded by the BEST1 gene, is a Ca2+-activated Cl- ...

Multi-target Parallel Processing Approach for Gene-to-structure Determination of the Influenza Polymerase PB2 Subunit

Pandemic outbreaks of highly virulent influenza strains can cause widespread morbidity and mortality in human populations worldwide. In the United States alone, an average of 41,400 deaths and 1.86 million hospitalizations are caused by influenza virus infection each year 1. Point mutations in the polymerase basic protein 2 subunit (PB2) have been linked to the adaptation of the viral infection in humans 2. Findings from such studies have revealed the biological significance of PB2 as a virulence factor, thus highlighting its potential as an antiviral drug target.
The structural genomics program put forth by the National Institute of Allergy and Infectious Disease (NIAID) provides funding to Emerald Bio and three other Pacific Northwest institutions that together make up the Seattle Structural Genomics Center for Infectious Disease (SSGCID). The SSGCID is dedicated to providing the scientific community with three-dimensional protein structures of NIAID category A-C pathogens. Making such structural information available to the scientific community serves to accelerate structure-based drug design.
Structure-based drug design plays an important role in drug development. Pursuing multiple targets in parallel greatly increases the chance of success for new lead discovery by targeting a pathway or an entire protein family. Emerald Bio has developed a high-throughput, multi-target parallel processing pipeline (MTPP) for gene-to-structure determination to support the consortium. Here we describe the protocols used to determine the structure of the PB2 subunit from four different influenza A strains.

Structure-based drug design plays an important role in drug development. Pursuing multiple targets in parallel greatly increases the chance of success for lead discovery. The following article highlights how the Seattle Structural Genomics Center for Infectious Disease utilizes a multi-target approach for gene-to-structure determination of the PB2 influenza A subunit.

流感聚合酶PB2亚基基因结构测定多目标并行处理方法

Pandemic outbreaks of highly virulent influenza strains can cause widespread morbidity and mortality in human populations worldwide. In the United States ...

免疫与感染

Engineering Cell-permeable Protein

The protein transduction technique enables the direct delivery of biologically active material into mammalian cells [for review see 1,2]. For this one can make use of the translocating ability of so-called cell penetrating peptides (CPPs), also designated as protein transduction domains (PTDs). The TAT-CPP derived from the human immunodeficiency virus type 1 (HIV-1) Tat (trans-activator of transcription) protein has been widely used. The positively charged TAT promotes cell permeability thereby overcoming the barriers of the cellular membrane by endocytosis or/and direct membrane penetration2. In combination with a nuclear localization signal (NLS) fusion proteins are able to enter the nucleus exhibiting functionality. Our video presentation demonstrates, as an exemplification for the engineering of cell-permeable proteins, the construction, production and application of a cell-permeable version of the DNA-modifying enzyme Cre.
Cre is a site-specific recombinase that is able to recognize and recombine 34 base pair loxP sites in mammalian cells in vitro and in vivo. Therefore the Cre/loxP system is widely used to conditionally induce mutations in the genome of living cells3,4. The delivery of active Cre recombinase to cells, however, represents a limitation.
We describe the pSESAME vector system, which allows a direct insertion of the gene-of-interest and provides a platform to rapidly clone different domains and tags used within the vector in a convenient and standardized manner. Rearranging of the different tags has been shown to modify the biochemical properties of the fusion proteins providing a possibility to achieve higher yield and better solubility. We demonstrate how to express and purify recombinant cell-permeant proteins in and from E. coli. The functionality of the recombinant Cre protein is finally validated in cell culture by assessing its intracellular recombinase activity.

Protein transduction enables the direct delivery of biologically active proteins into cells. In contrast to conventional methods such as DNA transfection or viral transduction this non-invasive paradigm allows highly efficient cellular manipulation in a titratable manner circumventing cellular toxicity and the risk of oncogenic transformation by permanent genetic modification.

工程细胞透水蛋白

The protein transduction technique enables the direct delivery of biologically active material into mammalian cells [for review see 1,2]. For this one can ...

生物学

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. ...

脂质双细胞中 ABC 转运蛋白膜蛋白的结晶

ABCG5/G8 Crystallization in a Lipidic Bicelle Environment for X-Ray Crystallography

ATP-binding cassette (ABC) transporters constitute lipid-embedded membrane proteins. Extracting these membrane proteins from the lipid bilayer to an aqueous environment is typically achieved by employing detergents. These detergents disintegrate the lipid bilayer and solubilize the proteins. The intrinsic habitat of membrane proteins within the lipid bilayer poses a challenge in maintaining their stability and uniformity in solution for structural characterization. Bicelles, which comprise a blend of long and short-chain phospholipids and detergents, replicate the natural lipid structure. The utilization of lipid bicelles and detergents serves as a suitable model system for obtaining high-quality diffraction crystals, specifically to determine the high-resolution structure of membrane proteins. Through these synthetic microenvironments, membrane proteins preserve their native conformation and functionality, facilitating the formation of three-dimensional crystals. In this approach, the detergent-solubilized heterodimeric ABCG5/G8 was reintegrated into DMPC/CHAPSO bicelles, supplemented with cholesterol. This setup was employed in the vapor diffusion experimental procedure for protein crystallization.

作者聚焦：用于 ABC 转运蛋白转运蛋白的 Bicelle 结晶设置以推进药物开发 

This protocol describes a setup for the crystallization of the sterol transporter ABCG5/G8. ABCG5/G8 is reconstituted into bicelles for hanging-drop crystallization. The protocol does not require specialized materials or substrates, making it accessible and easy to adapt in any laboratory for determining the protein structure through X-ray crystallography.

用于 X 射线晶体学的脂质双铈环境中的 ABCG5/G8 结晶

ATP-binding cassette (ABC) transporters constitute lipid-embedded membrane proteins. Extracting these membrane proteins from the lipid bilayer to an ...

表达和纯化溶质载体转运蛋白的策略

High-Throughput Expression and Purification of Human Solute Carriers for Structural and Biochemical Studies

Solute carriers (SLCs) are membrane transporters that import and export a range of endogenous and exogenous substrates, including ions, nutrients, metabolites, neurotransmitters, and pharmaceuticals. Despite having emerged as attractive therapeutic targets and markers of disease, this group of proteins is still relatively underdrugged by current pharmaceuticals. Drug discovery projects for these transporters are impeded by limited structural, functional, and physiological knowledge, ultimately due to the difficulties in the expression and purification of this class of membrane-embedded proteins. Here, we demonstrate methods to obtain high-purity, milligram quantities of human SLC transporter proteins using&#160;codon-optimized gene sequences. In conjunction with a systematic exploration of construct design and high-throughput expression, these protocols ensure the preservation of the structural integrity and biochemical activity of the target proteins. We also highlight critical steps in the eukaryotic cell expression, affinity purification, and size-exclusion chromatography of these proteins. Ultimately, this workflow yields pure, functionally active, and stable protein preparations suitable for high-resolution structure determination, transport studies, small-molecule engagement assays, and high-throughput in vitro screening.

作者聚焦:使用密码子优化基因表达和纯化人溶质载体转运蛋白 

Structural and biochemical studies of human membrane transporters require milligram quantities of stable, intact, and homogeneous protein. Here we describe scalable methods to screen, express, and purify human solute carrier transporters using codon-optimized genes.

重组蛋白表达，结晶和生物物理研究

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生产，结晶和结构测定

蛋白晶体的微结晶和

哺乳动物 Bestrophin 离子通道的表达与纯化

流感聚合酶PB2亚基基因结构测定多目标并行处理方法

工程细胞透水蛋白

使用巴库洛病毒表达载体系统生产重组PRMT蛋白

用于 X 射线晶体学的脂质双铈环境中的 ABCG5/G8 结晶

用于 X 射线晶体学的脂质双铈环境中的 ABCG5/G8 结晶

用于 X 射线晶体学的脂质双铈环境中的 ABCG5/G8 结晶

用于结构和生化研究的人溶质载体的高通量表达和纯化