Name: residue-specific incorporation of noncanonical amino acids into model proteins using an escherichia coli cell-free transcription-translation system
Uploaded: 2016-08-01T15:00:00.000+00:00
Duration: 11 min 47 s
Description: Watch this Scientific Journal Video about Residue-specific Incorporation of Noncanonical Amino Acids into Model Proteins Using an Escherichia coli Cell-free Transcription-translation System at JoVE.co

E.G. Worst and A. Ott acknowledge financial support by the Deutsche Forschungsgemeinschaft (DFG) within the collaborative research center SFB 1027 as well as Saarland University. E.G. Worst, A. Ott and V. Noireaux further acknowledge financial aid by the Human Frontiers Science Program Organization (HFSPO). The authors thank Tobias Baumann and Stefan Oehm (Institute of Chemistry, Technische Universit&#228;t Berlin) for critical reading.

The authors have nothing to disclose.

安易于使用的无细胞表达系统作为一个可行的策略，去渣特异性结合NCAA锦标赛成蛋白质，提出。为此，将粗提取物补充有载体DNA编码所关注的相应氨基酸的蛋白质，能量缓冲器和。需要注意的是粗提取物等分试样体积取决于粗提取物的蛋白质浓度34。无细胞表达效率取决于载体DNA构建浓度优化。能量缓冲器组件的体积为优化MG-和K-谷氨酸浓度的函数，以使所述无细胞表达模型蛋白的高产率而有所不同。 掺入实验的初步评估可以通过执行未纯化无细胞反应介质的SDS-PAGE来获得。有关更详尽的分析，HPLC-ESI质谱分析，提出为手段，以检查是否有完整的，具体的残留INCOR在NCAA的穿孔。至于后者的制备，旋转柱系统用于使His标签纯化和缓冲与我们在这个协议中使用小体积交换。 包括HPLC-ESI质谱，整个协议可在2天内进行。它不包括任何特别关键的步骤。然而，MG-和K-谷氨酸浓度的优化以及载体的DNA是为了表达的模型蛋白的高产率是至关重要的。使用高效表达载体的个体极值-OR 2-OR 1-PR-UTR1-gene_of_model_protein-T500强烈推荐。 His-标记的蛋白的洗脱通常是由于高浓度的咪唑（&gt; 150毫米）和其他盐如NaH 2 PO 4（&gt; 300毫米）或氯化钠（&gt; 50毫米）产生在质谱分析49高背景噪声。用合适的蛋白存储缓冲器这样的洗脱缓冲液的交换稳定模型prote在和大幅降低质谱分析中的背景噪声。 其结果是，可在模型蛋白质内的所有六个位置替换精氨酸。在表达系统中，没有精氨酸残基可以被检测出来。这简化相比，需要进一步消耗策略29,30其他表达系统精氨酸类似物的特定残基掺入。所提出的无细胞的方法规避那些因灿毒性在体内的方法存在固有局限性，或在单一蛋白生产策略24,31 mRNA序列的强烈依赖。相反， 在体外系统中采用，体内灿裂解丝氨酸和羟基胍确实发生31。 但是，无细胞系统保留足够量的Lys与类似物如HYL竞争。液相色谱 - ESI质谱分析表明，该模型蛋白含有两者，的Canonical以及不同比例的非规范类似物。赖氨酸的特定残基掺入是在一般可能的，但对于完全取代，进一步消耗策略，或特别设计的aaRS和tRNA的识别NCAA锦标赛的优化需要开发。 我们在相同的浓度为规范那些加入NCAA获得的无细胞表达，修饰的模型蛋白的优良的产率。掺入效率取决于NCAA的性质被并入。甚至更高的收益率可能仍然是通过优化NCAA的浓度实现的。 只要它们是由典型的内源性翻译系统接受所呈现的结果证明了NCAA锦标赛的特定残基掺入所采用的系统的适用性。对于特定NCAA锦标赛的特定残基掺入，一个另外的需求，以检查是否类似CAA distur的残B中的表达系统。 无细胞转录-翻译系统可以从不同的生物体进行工程化，以不同的需求54作出响应。所有E.在这里提出的无细胞系统的大肠杆菌转录-翻译机器使噬菌体和大肠杆菌的使用大肠杆菌的启动子，它们可以并行地或连续地在级联55采取行动。的普遍适用性和可用性使该方法在氨基酸毒性和治疗应用的进一步研究的有力工具。

遗传密码是通用的生物圈。它的代码的一组20种经典氨基酸，有时由硒代1或吡咯2延长。这是转换的帮助的tRNA的成折叠成蛋白质的氨基酸链的遗传密码的核糖体。规范氨基酸的官能团，与翻译后修饰组合，向一个非常广泛的蛋白质功能3,4。原则上，由于该组有限的经典氨基酸功能限制可以通过进一步结合来克服，非规范氨基酸（NCAA锦标赛），使新的化学和新的功能3,4-。 还有的NCAA锦标赛的结合两种互补的方法:在定点或特定渣掺入。前一种方法需要大量的技术难题，因为规范的一套氨酰-tRNA合成器的etases（的aaRS）和tRNA的必须由正交的aaRS酰tRNA对，必须不与内源性翻译机制相互作用进行扩展。基于仔细的工程，这种方法采用了NCAA锦标赛截至所需的蛋白质站点的单点突变。 NCAA锦标赛的位点特异性掺入基因由未分配给典型氨基酸（CAA），通常是一个终止密码子5-9的密码子编码。这种方法需要在给定的网站在功能上的变化，而 ​​不是整个蛋白10-13。 相反，具体的残基掺入依赖于规范翻译机器错误识别的非规范氨基酸。掺入的发生是由于缺乏的aaRS的底物特异性。 NCAA锦标赛的特定残基掺入，建立在科恩和同事14的工作，导致了重要的应用3,10，其中蛋白质的生物正交标记15-17或X射线晶体18蛋白质的结构解析。 由于天然的aaRS一般都喜欢在一个同构NCAA其同源的氨基酸，体内特定渣掺入效率通常需要营养缺陷型表达宿主不能合成NCAA的规范模拟。的宿主细胞培养在生长培养基中，可提供仅类似CAA的低浓度。它与补充的连续与NCAA组合枯竭迫使表达宿主纳入了NCAA到模型中的蛋白质在多个，规范地规定站点。与此相反的位点特异性的方式，这通常对整个蛋白结构的深的影响，从而导致相当修饰的蛋白质19,20的物理和化学性质。然而，大多数的NCAA锦标赛的是表达宿主3生长抑制剂，因为它们被结合到许多其他PROT除了重组基因表达过程中的利益EINS。这清楚地限制了体内的方法。的氨基酸，是有毒或对蛋白质结构强的影响的体内掺入仍然特别具有挑战性。然而，这些分子的工程师具有非凡的功能的蛋白质中最有前途的。 一个例子是有毒的，非规范，天然存在的L-刀豆氨酸（CAN），L-精氨酸（Arg）的类似物。它影响和块ARG有关的调节和催化反应途径，以及其在活细胞的存在可以导致立即死亡3,21-23。其纳入蛋白质精氨酸在位置可以减少蛋白质的稳定性21-23。由于所得的毒性，含有在大肠杆菌 （ 大肠杆菌 ）和其它普通的表达宿主的蛋白质刀豆氨酸的表达仍然是一个挑战。由于这些原因，完整的体内我灿ncorporation在所有的精氨酸位置已经恰当地被证实只有一次24，使用单阐述蛋白质的生产体系。然而，也已经被提出作为一种抗癌剂25-27，并作为用于人28自身免疫性疾病的刺激。此外，它受到在其抗代谢，抗细菌，抗真菌的各种研究和抗病毒特性25的。这些特性提高了需求高效，​​易于执行的方法来表达可含有蛋白质为药用，医学和功能研究。 虽然连接到体内生产的许多问题可以使用无细胞表达系统来回避， 在体外特异性残基的办法只被很差探讨。已报道的L-色氨酸类似物29和多个NCAA锦标赛30的无细胞特异性残基结合。这些方法是基于高度efficient T7 RNA聚合酶。 T7 RNA聚合酶嗣继承噬菌体样转录，从而减少在比较源性转录遗传功能。 罐的完整特定残基掺入模型蛋白在所有精氨酸位置最近报道31，使用无细胞表达系统32。同一系统的一个稍微的改变通过停止启用不同的吡咯类似物的位点特异性并入模型蛋白抑制密码子33。所采用的无细胞系统31 - 33是基于一个所有大肠杆菌大肠杆菌转录-翻译系统。然而，它使蛋白表达尽可能有效电流噬菌体系统（0.5 -为1mg / ml重组蛋白）32，同时保留大部分的原始转录-翻译模块化。 在这项工作中，一个详细的协议被提供关于如何渣油NCAA锦标赛的UE特定掺入可实现，使用此所有大肠杆菌大肠杆菌无细胞系统32。此外，提出进一步的措施，准备通过HPLC-ESI质谱恰当的评价表达的蛋白质。为了扩大这种无细胞系统的特性，这项工作并不仅仅指灿31日公布的合并，但也提出相关的非经典的L-赖氨酸模拟L-羟基赖氨酸新的数据。 为NCAA锦标赛的特定残基掺入下列协议是最近发表在朱庇特34的协议的适应。后者协议描述如何执行与标准氨基酸高效无细胞表达。此外，它提出了粗无细胞提取物的制备中，氨基酸溶液，能量储备溶液，并在该方法中使用的能量缓冲器。以下方案相比集中在改性步骤将前面Protocol为了使NCAA锦标赛的特定残基掺入。校准的移液管，低结合枪头和微离心管建议的准备。在下面，使用IUPAC缩写为氨基酸。

<table><tbody><tr><td>Protective eyewear</td><td>Sigma-Aldrich, St. Louis, USA</td><td>Z758841</td><td></td></tr><tr><td>Nitrile gloves (size S)</td><td>Sigma-Aldrich, St. Louis, USA</td><td>Z768960</td><td>Catalog numbers other sizes: Z768979 for M, Z768987 for L and Z768995 for XL</td></tr><tr><td>Eppendorf Safe-Lock Tube 1.5 ml (PCR clean)</td><td>Eppendorf, Hamburg, Germany</td><td>30123.328</td><td></td></tr><tr><td>Microbalance Discovery DV114CM</td><td>Ohaus, Greifensee, Switzerland</td><td>80104140</td><td></td></tr><tr><td>Microspatula (L 6 5/8 in., stainless steel, rod diam. 0.09 in.)</td><td>Sigma-Aldrich, St. Louis, USA</td><td>Z243213</td><td></td></tr><tr><td>L-Canavanine</td><td>Sigma-Aldrich, St. Louis, USA</td><td>C9758</td><td>Acute toxicity: wear eyeshields, dust mask, protective gloves</td></tr><tr><td>Hydroxylysine (racemic mixture)</td><td>Sigma-Aldrich, St. Louis, USA</td><td>H0377</td><td></td></tr><tr><td>Cryo-gloves (size S, water resistent)</td><td>Sigma-Aldrich, St. Louis, USA</td><td>Z183490</td><td>Catalog numbers other sizes: Z183512 for M, Z183520 for L and Z183539 for XL</td></tr><tr><td>RTS Amino Acid Sampler</td><td>Biotechrabbit, Hennigsdorf, Germany</td><td>BR1401801</td><td>For homemade preparation of amino acid stock solutions, follow this protocol&lt;sup&gt;35&lt;/sup&gt; and use the solid amino acid kit LAA21-1KT, L-proline (81709-25G), L-cysteine (30089-25G), L-histidine (53319-25G) and L-lysine (L5501-5G)&amp;nbsp; (all from Sigma-Aldrich, St. Louis, USA)</td></tr><tr><td>HEPES</td><td>Sigma-Aldrich, St. Louis, USA</td><td>H6147</td><td></td></tr><tr><td>ATP</td><td>Sigma-Aldrich, St. Louis, USA</td><td>A8937</td><td></td></tr><tr><td>CTP</td><td>Affymetrix, Santa Clara, USA</td><td>14121</td><td></td></tr><tr><td>GTP</td><td>Affymetrix, Santa Clara, USA</td><td>16800</td><td></td></tr><tr><td>UTP</td><td>Affymetrix, Santa Clara, USA</td><td>23160</td><td></td></tr><tr><td>tRNA (from &lt;em&gt;E. coli&lt;/em&gt;, pack size 100 mg)</td><td>Sigma-Aldrich, St. Louis, USA</td><td>10109541001</td><td>Catalog number for pack size of 500 mg is 10109550001</td></tr><tr><td>CoA</td><td>Sigma-Aldrich, St. Louis, USA</td><td>C4282</td><td></td></tr><tr><td>NAD (from yeast )</td><td>Sigma-Aldrich, St. Louis, USA</td><td>N6522</td><td></td></tr><tr><td>cAMP</td><td>Sigma-Aldrich, St. Louis, USA</td><td>A9501&amp;nbsp;</td><td></td></tr><tr><td>Folinic acid</td><td>Sigma-Aldrich, St. Louis, USA</td><td>F7878</td><td></td></tr><tr><td>3-PGA</td><td>Sigma-Aldrich, St. Louis, USA</td><td>P8877</td><td></td></tr><tr><td>Mg-glutamate</td><td>Sigma-Aldrich, St. Louis, USA</td><td>49605</td><td></td></tr><tr><td>K-glutamate</td><td>Sigma-Aldrich, St. Louis, USA</td><td>G1149</td><td></td></tr><tr><td>pBEST-OR2-OR1-Pr-UTR1-deGFP-T500</td><td>Addgene, Cambridge, USA</td><td>Plasmid #40019</td><td></td></tr><tr><td>4-20% precast Tris-Glycine Gels (10 cm x 10 cm x 1 mm, 10 courses)</td><td>Anamed Elektrophorese, Gro&amp;szlig;-Bieberau / Rodau, Germany</td><td>TG 81610</td><td></td></tr><tr><td>SDS running buffer (10 x concentrate, 5,000 ml)</td><td>Anamed Elektrophorese, Gro&amp;szlig;-Bieberau / Rodau, Germany</td><td>TG 50001</td><td></td></tr><tr><td>SDS loading buffer (2 x concentrate, 50 ml)</td><td>Anamed Elektrophorese, Gro&amp;szlig;-Bieberau / Rodau, Germany</td><td>TG 05002</td><td></td></tr><tr><td>Unstained protein marker, broad range (2-212 kDa)</td><td>New England Biolabs, Ipswich, USA</td><td>P7702S</td><td></td></tr><tr><td>Methanol</td><td>Merck, Darmstadt, Germany</td><td>1060091011</td><td>Toxic by inhalation, in contact with skin and if swallowed: wear protective gloves and work under fume hood</td></tr><tr><td>Acetic acid (99.8%)</td><td>VWR International, Darmstadt, Germany</td><td>20104.447</td><td></td></tr><tr><td>Coomassie Blue G-250 (10 g)</td><td>Biozym Scientific, Hessisch Oldendorf, Germany</td><td>902120</td><td></td></tr><tr><td>His-Spin Protein Miniprep kit</td><td>Zymo Research Europe, Freiburg, Germany</td><td>P2002</td><td>Product also distributed by Zymo Research Corporation, Irvine, USA&amp;nbsp;</td></tr><tr><td>Trizma Base</td><td>Sigma-Aldrich, St. Louis, USA</td><td>T1503</td><td></td></tr><tr><td>Hydrochloric acid</td><td>Sigma-Aldrich, St. Louis, USA</td><td>H1758</td><td></td></tr><tr><td>Glycerol, 99%</td><td>VWR International, Darmstadt, Germany</td><td>24397.296DB</td><td></td></tr><tr><td>CentriPure Z25 mini spin columns</td><td>Genaxxon bioscience, Ulm, Germany</td><td>CP-0205-Z100</td><td></td></tr><tr><td>Sodium chloride</td><td>Sigma-Aldrich, St. Louis, USA</td><td>S9888</td><td></td></tr><tr><td>Concentrator 5301</td><td>Eppendorf, Hamburg, Germany</td><td>5301 000.210</td><td></td></tr><tr><td>2xYT</td><td>MP biomedicals, Santa Ana, USA</td><td>113012032</td><td></td></tr><tr><td>Bacto-Agar</td><td>BD Diagnostics, Franklin Lakes, USA</td><td>214010</td><td></td></tr><tr><td>Bead-beating tubes (polypropylene microvials)</td><td>Biospec, Bartlesville, USA</td><td>522S</td><td></td></tr><tr><td>Beads, 0.1&amp;nbsp;mm dia.</td><td>Biospec, Bartlesville, USA</td><td>11079101</td><td></td></tr><tr><td>BL21 Rosetta 2 &lt;em&gt;E. coli&lt;/em&gt; strain</td><td>Merck, Darmstadt, Germany</td><td>71402</td><td></td></tr><tr><td>Bradford BSA protein assay Kit</td><td>Bio-Rad, M&amp;uuml;nchen, Germany</td><td>500-0201</td><td></td></tr><tr><td>Chloramphenicol</td><td>Sigma-Aldrich, St. Louis, USA</td><td>C1919</td><td></td></tr><tr><td>Cuvettes, 1.5&amp;nbsp;ml</td><td>Thermo Fisher Scientific, Waltham, USA</td><td>14-955-127</td><td></td></tr><tr><td>DTT</td><td>Sigma-Aldrich, St. Louis, USA</td><td>D0632</td><td></td></tr><tr><td>Micro Bio-Spin Chromatography Columns</td><td>Bio-Rad, M&amp;uuml;nchen, Germany</td><td>732-6204</td><td></td></tr><tr><td>Nunc 384-well optical bottom plates</td><td>Thermo Fisher Scientific, Waltham, USA</td><td>142761</td><td></td></tr><tr><td>Nunc sealing tape</td><td>Thermo Fisher Scientific, Waltham, USA</td><td>232701</td><td></td></tr><tr><td>PEG-8000</td><td>Promega, Madison, USA</td><td>V3011</td><td></td></tr><tr><td>Potassium phosphate dibasic solution</td><td>Sigma-Aldrich, St. Louis, USA</td><td>P8584</td><td></td></tr><tr><td>Potassium phosphate monobasic solution</td><td>Sigma-Aldrich, St. Louis, USA</td><td>P8709</td><td></td></tr><tr><td>Slide-A-Lyzer dialysis cassettes, 10k MWCO (Kit)</td><td>Thermo Fisher Scientific, Waltham, USA</td><td>66382</td><td></td></tr><tr><td>Spermidine</td><td>Sigma-Aldrich, St. Louis, USA</td><td>85558</td><td></td></tr><tr><td>1&amp;nbsp;L centrifuge bottle</td><td>Beckman-Coulter, Brea, USA</td><td>A98813</td><td></td></tr><tr><td>4&amp;nbsp;L Erlenmeyer flask</td><td>Kimble Chase, Vineland (NJ), USA</td><td>26500-4000</td><td></td></tr><tr><td>Avanti J-26XP centrifuge</td><td>Beckman-Coulter, Brea, USA</td><td>393127</td><td>Or equivalent centrifuge able to centrifuge 1 L bottles.</td></tr><tr><td>Forma 480 orbital shaker</td><td>Thermo Fisher Scientific, Waltham, USA</td><td>480</td><td>Or equivalent shaker able to shake chest-size 6 x 4 L .</td></tr><tr><td>JLA-8.1000 rotor</td><td>Beckman-Coulter, Brea, USA</td><td>363688</td><td>Or equivalent 5,000 x g rotor for the centrifuge above, able to centrifuge 1 L bottles.</td></tr><tr><td>Mini-Beadbeater-1</td><td>Biospec, Bartlesville, USA</td><td>3110BX</td><td></td></tr><tr><td>Microfuge 22R refrigerated microcentrifuge</td><td>Beckman-Coulter, Brea, USA</td><td>368831</td><td>Or equivalent centrifuge able to centrifuge 2 ml reaction tubes.</td></tr><tr><td>Heating block HLC HBT 130</td><td>Labexchange, Burladingen, Germany</td><td>24465</td><td>Or equivalent heating block able to heat samples in reaction tubes up to 100 &amp;deg;C.</td></tr><tr><td>Eppendorf MiniSpin centrifuge</td><td>Eppendorf, Hamburg, Germany</td><td>5452000018</td><td>Or equivalent centrifuge able to centrifuge 2 ml reaction tubes.</td></tr><tr><td>IKA Vortex 3 (4 mm orbital shaker diameter, 0 - 2,500 rpm)</td><td>Sigma-Aldrich, St. Louis, USA</td><td>Z654760</td><td>Or equivalent vortex.</td></tr><tr><td>Scotsman AF103 ice flaker machine</td><td>K&amp;auml;lte-Berlin, Berlin, Germany</td><td>AF103</td><td>Or equivalent ice flaker machine.</td></tr><tr><td>MyTemp mini digital incubator</td><td>Sigma-Aldrich, St. Louis, USA</td><td>Z763314</td><td>Or equivalent incubator able to heat samples at 29 &amp;deg;C.</td></tr><tr><td>EcoCell electrophoresis cell / chamber</td><td>Anamed Elektrophorese, Gro&amp;szlig;-Bieberau / Rodau, Germany</td><td>AN12005</td><td>Or equivalent electrophoresis chamber able to perform vertical gel electrophoresis with the above precast gels or other gels used.</td></tr><tr><td>Power-phor power supply for electrophoresis cell / chamber</td><td>Anamed Elektrophorese, Gro&amp;szlig;-Bieberau / Rodau, Germany</td><td>AN12001</td><td>Or equivalent power supply able to supply above used electrophoresis cell / chamber with power</td></tr><tr><td>VWR Signature Ergonomic High Performance Single-Channel Variable Volume Pipettors Starter Kit 1</td><td>VWR International, Darmstadt, Germany</td><td>613-5278</td><td>Or equivalent micropipettes enabling to pipette similar volumes with the same precision</td></tr><tr><td>VWR Signature Ergonomic High Performance Single-Channel Variable Volume Pipettors Starter Kit 2</td><td>VWR International, Darmstadt, Germany</td><td>613-5279</td><td>Or equivalent micropipettes enabling to pipette similar volumes with the same precision</td></tr><tr><td>Pipetman Tips Diamond D10ST (0.1 - 10 &amp;micro;l)</td><td>Gilson, Middleton, USA</td><td>F171101</td><td>Or equivalent low-binding pipette tips enabling to pipette similar volumes with the same precision</td></tr><tr><td>Pipetman Tips Diamond D200ST (2 - 200 &amp;micro;l)</td><td>Gilson, Middleton, USA</td><td>F171301</td><td>Or equivalent low-binding pipette tips enabling to pipette similar volumes with the same precision</td></tr><tr><td>Pipetman Tips Diamond D1000ST (100 - 1,000&amp;nbsp;&amp;micro;l)</td><td>Gilson, Middleton, USA</td><td>F171501</td><td>Or equivalent low-binding pipette tips enabling to pipette similar volumes with the same precision</td></tr><tr><td>50 ml centrifuge tubes with screw caps (sterile)</td><td>VWR International, Darmstadt, Germany</td><td>50-0156</td><td></td></tr><tr><td>15 ml centrifuge tubes with screw caps (sterile)</td><td>VWR International, Darmstadt, Germany</td><td>525-0150</td><td></td></tr><tr><td>14 ml polypropylene tubes (round bottom, two-position vent stopper, sterile)</td><td>Greiner Bio-One, Frickenhausen, Germany</td><td>187262</td><td></td></tr><tr><td>Discovery BIO Wide Pore C5 HPLC Column (3 &amp;micro;m particle size, L x I.D. 10 cm x 2.1 mm)</td><td>Sigma-Aldrich, St. Louis, USA</td><td>567227-U</td><td></td></tr><tr><td>Agilent 1260 HPLC machine</td><td>Agilent Technologies, Santa Clara, USA</td><td>G1312B</td><td></td></tr><tr><td>6500 Series Accurate-Mass Quadrupole Time-of-Flight (Q-TOF) LC/MS</td><td>Agilent Technologies, Santa Clara, USA</td><td>G6530BA</td><td></td></tr><tr><td>Acetonitrile&amp;nbsp;</td><td>Sigma-Aldrich, St. Louis, USA</td><td>270717</td><td></td></tr><tr><td>FLUOstar Omega microplate reader</td><td>BMG Labtech, Ortenberg, Germany</td><td>415-101</td><td>Or equivalent microplate reader able to measure the fluorescence of the expressed model protein</td></tr><tr><td>Hanna Checker pH meter</td><td>Sigma-Aldrich, St. Louis, USA</td><td>Z351091&amp;nbsp;</td><td></td></tr><tr><td>Formic acid eluent additive for LC-MS</td><td>Sigma-Aldrich, St. Louis, USA</td><td>56302</td><td></td></tr></tbody></table>

residue-specific incorporation of noncanonical amino acids into model proteins using an escherichia coli cell-free transcription-translation system

的标准集氨基酸导致了一个非常广泛的蛋白质的功能。然而，该组的残基仍对潜在的蛋白的应用的限制。非规范的氨基酸的结合可以扩大这个范围。有用于非规范氨基酸结合两种互补的方法。为位点特异性掺入，除了内源性的规范平移机械，正交氨酰基-tRNA合成酶氨酰tRNA对必须提供不与规范那些交互。因此，未分配到典型的氨基酸，通常是一个终止密码子的密码子，也是必需的。此遗传密码膨胀使非经典氨基酸的掺入在蛋白质内一个单一的，给定的位点。在这里提出的工作描述了特定残基掺入，其中遗传密码是内源性的翻译系统内重新分配。翻译机器一ccepts的非规范氨基酸作为替代在标准地规定的地点， 即掺入它，在该蛋白质的典型氨基酸出现的所有被非规范取代。的非规范氨基酸掺入可以改变蛋白质的结构，从而引起相当改性的物理和化学性质。非规范氨基酸类似物常常充当细胞生长抑制剂表达宿主，因为它们修改内源性蛋白， 在体内蛋白质生产限制性的。 在体内的毒性非规范氨基酸掺入蛋白质仍然特别具有挑战性。这里，对于由非经典氨基酸L-刀豆氨酸的完全替代L-精氨酸的无细胞的方法，提出。它绕过体内表达的固有的困难。此外，包括准备靶蛋白进行质谱分析的协议。它表明，L-赖氨酸可以通过将L-羟基赖氨酸取代，尽管具有较低的效率。原则上，任何非规范氨基酸类似物可以使用所提出的方法，只要内源性体外翻译系统识别它被并入。

此协议通过NCAA锦标赛的无细胞特异性残基掺入模型蛋白质指导。它提出了纳入实验，并进一步步骤准备模型蛋白质的适当HPLC-ESI质谱分析的初步评估SDS-PAGE。 这里，精氨酸类似物可以的无细胞特异性残基掺入，以及赖氨酸模拟L - 羟基赖氨酸（HYL）的代表性结果。不同的氨基酸溶液，能量缓冲器，如上所述制备的模型蛋白和无细胞反应的载体DNA编码。参考无细胞反应被提供有由所述20中国农业科学院的氨基酸溶液。对于每一个实验中，阴性对照无细胞反应用缺乏在NCAA的问题的典型的模拟的氨基酸溶液供给。对于每个APPRoach一无细胞反应表达在氨基酸溶液，其中该CAA是由非规范模拟取代的存在下模型蛋白。 His标签纯化，缓冲液交换和HPLC-ESI质谱分析是按照上述的协议执行。 该模型蛋白是C-末端His标签的deGFP 32，EGFP 53的截短形式。其单字母氨基酸序列可以在（补充材料2）中找到。该模型蛋白含有6 Arg和18赖氨酸的位置， 分别表达载体是的pbest-OR 2-OR 1-PR-UTR1-deGFP-T500。 在NCAA的完全结合的情况下，可以假设的阴性对照反应不表达deGFP，由于20 CAAS缺失中的一个。相反，deGFP必须在其它两个反应检测:在refe天然1伦斯无细胞反应，并在被提供与NCAA无细胞反应修饰蛋白。 图1A示出了制罐掺入实验的初步的SDS-PAGE评估。参考无细胞反应具有最高deGFP表达水平。在被设置有可在无细胞反应，deGFP在稍低浓度表示。没有deGFP表达所用的阴性对照来检测。此的SDS-PAGE的结果是对罐的一个成功的掺入靶蛋白deGFP的良好指示。 为了证明制罐的假设完整纳入deGFP，既纯化模型蛋白质，在图1B可见，通过HPLC-ESI质谱进行分析。 图1C示出了纯化的deGFP分子的解卷积质谱。 deGF的解卷积质量<html>即在参考无细胞反应表示P是26,192.8道尔顿。对于deGFP在含有无细胞反应出现26202.5大的质量的制罐表达。对于本地deGFP6Arg和精氨酸修饰deGFP6Can预期由群众在被全面取代是26193 Da和26204大分别。 1.5大的为deGFP6Can的质量差是光谱去卷积的误差范围内。因此，能充分纳入deGFP在所有6精氨酸位置被确认。 减少强度的两个峰值对应于天然deGFP6Arg和改性deGFP6Can未达到其成熟荧光团。荧光团是自催化通过消除一个水分子的产生，接着氧化。这导致了质量提高了20大，如果这个过程无法继续。 PG"/&gt; 图 1. 无细胞表达和纯化deGFP分子可以掺入实验和HPLC-ESI质谱的SDS-PAGE评估。（A）中使用SDS-PAGE的能掺入实验的初步评价。从左至右依次为:蛋白质标准，标准无细胞反应，阴性对照和无细胞反应提供可替代精氨酸。 His标签纯化后（B）的SDS-PAGE和缓冲表示deGFP分子交换。从左至右依次为:蛋白质标准，从参考反应提纯deGFP，由含灿反应纯化deGFP。 （C）采用HPLC-ESI质谱灿全部并入的确认。本地deGFP和精氨酸修饰deGFP的预期由群众完全可以取代是26193 Da和26204大分别。每个光谱被归一化到其最高强度（计数）。峰值位置指示在大。为可视化的目的，将凝胶泳道被从凝胶图片提取的，连接在一起，被转换成灰度格式，大小被优化和对比度以及亮度增强。原厂胶道在补充材料3介绍<a href="https://www.jove.com/files/ftp_upload/54273/54273fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> 图2A示出了HYL掺入实验的初步的SDS-PAGE评估。在于设置有HYL无细胞反应，deGFP表示。相反于第一实验中，将弱deGFP带所用的阴性对照反应中观察到。这可能是由于在无细胞反应赖氨酸残基。这使得在阴性对照反应，其中加入既不赖氨酸也不HYL微弱deGFP表达。 用于h<html> PLC-ESI质谱，设置有HYL无细胞反应的deGFP分子是纯化的和交换缓冲液（ 图2B）。 <img alt="图2" src="/files/ftp_upload/54273/54273fig2.jpg" /> 图2. SDS-PAGE中掺入HYL实验的评价 （A）从左至右依次为:阴性对照无细胞反应，含HYL和蛋白质的标准无细胞的反应。 His标签纯化后（B）的SDS-PAGE和缓冲表示deGFP分子交换。从左至右依次为:含反应和蛋白质纯化标准从deGFP的HYL。为可视化的目的，将凝胶泳道被从凝胶图片提取的，连接在一起，被转换成灰度格式，大小被优化和对比度以及亮度增强。原厂胶道在补充材料3中。F ="htt​​ps://www.jove.com/files/ftp_upload/54273/54273fig2large.jpg"目标="_空白"&gt;点击此处查看该图的放大版本。 图3示出纯化deGFP分子的解卷积质谱。 图3A证实在无细胞的反应已经存在赖氨酸残基的假说。频谱的主要峰对应于天然deGFP（预期质量:26193道尔顿）。再次，一个20达较高的质量未开发其荧光团deGFP分子可以被检测出来。的赖氨酸残基被优先加载到tRNA的赖氨酸由赖氨酰tRNA合成酶，导致天然deGFP18Lys的高表达水平。 HYL和Lys之间的质量差是16达。由于HYL的存在是在竞争的赖氨酸残基被生成的所有可能的deGFP物种（deGFP18Hyl，deGFP17Hly + 1Lys，...，deGFP16Hyl + 2Lys）（ 图3B）。诚然，deGFP1Hyl + 17Lys的峰值与没有产生其荧光（ 图3A）和一些峰的质谱与预期质量相差超过2达天然deGFP的峰值重叠。这些质量的差异可以归因于高噪音由于低量的deGFP物种。然而，HYL一般是由无细胞系统并入。进一步改进必须完成取消在无细胞反应赖氨酸残基。 <img alt="图3" src="/files/ftp_upload/54273/54273fig3.jpg" /> 图 3. HPLC-ESI质谱含有HYL无细胞反应的纯化deGFP分子光谱 （A）中本机deGFP18Lys主要是检测到（预期群众:用荧光团26193道尔顿，而不成熟荧光团26213道尔顿）。 （B）放大倍数显示所有可能deGFP种（deGFP18Hyl，deGFP17Hly + 1Lys，deGFP16Hyl + 2Lys，...，deGFP1Hyl + 17Lys）的存在。其预期群众26193大+ N×16大（N = 1，...，18）。频谱归一化到其最高强度（计数）。峰的位置以达表示。 <a href="https://www.jove.com/files/ftp_upload/54273/54273fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图1" src="/files/ftp_upload/54273/54273supfig1.jpg" /> 补充材料1.计算模板。在部分2中，能量缓冲器主混合物的制备是用最佳MG-30微升和粗提物的等分试样，并分别为3毫米的K-谷氨酸浓度和30mM的举例说明。这个例子导致了产生3个能量缓冲等分预混量。在秒化3，无细胞反应的制备采用90纳米DNA原液导致无细胞反应的10纳米的最佳载体DNA浓度例证。 <a href="https://www.jove.com/files/ftp_upload/54273/cell-free_calculations_JoVE_11.03.2016_final.xlsx" target="_blank">请点击此处下载此文件。</a> 粗提取物体积:28微升，最佳的Mg谷氨酸:2毫米，最佳的K-谷氨酸:40毫米，编号为适当的可追溯性，使用计算模板是由从上面的例子不同，这些典型值的插入例举的所需的缓冲区等份:100，在无细胞反应最佳矢量浓度:8纳米，DNA载体原液:150纳米。 首先进入28微升的粗提取物量进入第一次模板部分的橙色领域。然后，进入到第二模板部2mm的D 40毫米为最佳MG-和K-谷氨酸浓度为橙色领域。考虑到最佳MG-和K-浓度，15微升能量缓冲液的组成，以及一个相应的，按比例增加16微升等分试样被计算。下面，相应输入100作为能量缓冲器等分的期望数目（16微升）。模板适应为1,700微升主结构不同的缓冲部件的体积为:204微升100毫米镁谷氨酸原液，136微升3M的K-谷氨酸原液，728.73微升14倍的能源解决方案，510微升的40％PEG-8000和121.27微升无菌DDH 2 O最后，在第三模板部分，输入8 50nm和150nm作为无细胞反应最佳矢量浓度分别载体DNA原液浓度。模板适应必须被添加到28微升粗提取物的不同组分的量以完成一个90微升的制备无细胞反应如下:15微升能量缓冲液，15微升3不同组成的氨基酸溶液之一，4.80微升的载体DNA溶液（150纳米），和27.20微升无菌DDH 2 O的<img alt="图2" src="/files/ftp_upload/54273/54273supfig2.jpg" /> 补充材料2.一个字母的模型蛋白deGFP的氨基酸序列。这个模型蛋白含有6精氨酸和赖氨酸18立场。 <a href="https://www.jove.com/files/ftp_upload/54273/54273supfig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图3" src="/files/ftp_upload/54273/54273supfig3.jpg" /> 补充材料3，全长度和对应于该凝胶图片未修改凝胶泳道呈现图1和2中的凝胶泳道中的每个的各个S呈现<html> ubfigure由相同的SDS聚丙烯酰胺凝胶中提取。在图1和2 中这些泳道进行演示接合在一起。蛋白质标准带的分子量的数字旁边指出。 （A.1） 图1A的未裁剪胶道。从左至右依次为:蛋白质标准，标准无细胞反应，阴性对照和无细胞反应提供可替代精氨酸。 （A.2） 图1B的未裁剪胶道。从左至右依次为:蛋白质标准，从参考反应提纯deGFP，由含灿反应纯化deGFP。 （B.1） 图2A的未裁剪胶道。从左至右依次为:阴性对照无细胞反应，含HYL和蛋白质的标准无细胞的反应。 （B.2） 图2B的未裁剪胶道。从左至右依次为:含反应和蛋白质纯化标准从deGFP的HYL。D / 54273 / 54273supfig3large.jpg"目标="_空白"&gt;点击此处查看该图的放大版本。

Watch this Scientific Journal Video about Residue-specific Incorporation of Noncanonical Amino Acids into Model Proteins Using an Escherichia coli Cell-free Transcription-translation System at JoVE.co

Residue-specific Incorporation of Noncanonical Amino Acids into Model Proteins Using an Escherichia coli Cell-free Transcription-translation System

An easy-to-use, cell-free expression protocol for the residue-specific incorporation of noncanonical amino acid analogs into proteins, including downstream analysis, is presented for medical, pharmaceutic, structural and functional studies.

非经典氨基酸的具体残留纳入到模型蛋白质使用<em&gt;大肠杆菌</em&gt;无细胞转录翻译系统

The canonical set of amino acids leads to an exceptionally wide range of protein functionality. Nevertheless, the set of residues still imposes ...

residue-specific-incorporation-noncanonical-amino-acids-into-model

Research

JoVE Journal

Biology

Residue-specific Incorporation of Noncanonical Amino Acids into Model Proteins Using an Escherichia coli Cell-free Transcription-translation System

15.9K 次观看.  Saarland University. An easy-to-use, cell-free expression protocol for the residue-specific incorporation of noncanonical amino acid analogs into proteins, including downstream analysis, is presented for medical, pharmaceutic, structural and functional studies.

视频: Residue-specific Incorporation of Noncanonical Amino Acids into Model Proteins Using an Escherichia coli Cell-free Transcription-translation System

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

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

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

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

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

科研

化学

Escherichia coli-Based Cell-Free Protein Synthesis: Protocols for a robust, flexible, and accessible platform technology

Over the last 50 years, Cell-Free Protein Synthesis (CFPS) has emerged as a powerful technology to harness the transcriptional and translational capacity of cells within a test tube. By obviating the need to maintain the viability of the cell, and by eliminating the cellular barrier, CFPS has been foundational to emerging applications in biomanufacturing of traditionally challenging proteins, as well as applications in rapid prototyping for metabolic engineering, and functional genomics. Our methods for implementing an E. coli-based CFPS platform allow new users to access many of these applications. Here, we describe methods to prepare extract through the use of enriched media, baffled flasks, and a reproducible method of tunable sonication-based cell lysis. This extract can then be used for protein expression capable of producing 900 &#181;g/mL or more of super folder green fluorescent protein (sfGFP) in just 5 h from experimental setup to data analysis, given that appropriate reagent stocks have been prepared beforehand. The estimated startup cost of obtaining reagents is $4,500 which will sustain thousands of reactions at an estimated cost of $0.021 per &#181;g of protein produced or $0.019 per &#181;L of reaction. Additionally, the protein expression methods mirror the ease of the reaction setup seen in commercially available systems due to optimization of reagent pre-mixes, at a fraction of the cost. In order to enable the user to leverage the flexible nature of the CFPS platform for broad applications, we have identified a variety of aspects of the platform that can be tuned and optimized depending on the resources available and the protein expression outcomes desired.

Escherichia coli-Based Cell-Free Protein Synthesis: Protocols for a robust, flexible, and accessible platform technology

This protocol details the steps, costs, and equipment necessary to generate E. coli-based cell extracts and implement in vitro protein synthesis reactions within 4 days or less. To leverage the flexible nature of this platform for broad applications, we discuss reaction conditions that can be adapted and optimized.

大肠杆菌无细胞蛋白合成: 一种强大、灵活和可访问的平台技术的协议

Over the last 50 years, Cell-Free Protein Synthesis (CFPS) has emerged as a powerful technology to harness the transcriptional and translational capacity ...

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

Acetylated histone proteins can be easily expressed in Escherichia coli encoding a mutant, N&#949;-acetyl-lysine (AcK)-specific Methanosarcina mazi pyrrolysine tRNA-synthetase (MmAcKRS1) and its cognate tRNA (tRNAPyl) to assemble reconstituted mononucleosomes with site specific acetylated histones. MmAcKRS1 and tRNAPyl deliver AcK at an amber mutation site in the mRNA of choice during translation in Escherichia coli. This technique has been used extensively to incorporate AcK at H3 lysine sites. Pyrrolysyl-tRNA synthetase (PylRS) can also be easily evolved to incorporate other noncanonical amino acids (ncAAs) for site specific protein modification or functionalization. Here we detail a method to incorporate AcK using the MmAcKRS1 system into histone H3 and integrate acetylated H3 proteins into reconstituted mononucleosomes. Acetylated reconstituted mononucleosomes can be used in biochemical and binding assays, structure determination, and more. Obtaining modified mononucleosomes is crucial for designing experiments related to discovering new interactions and understanding epigenetics.

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

Here we present a method to express acetylated histone proteins using genetic code expansion and assemble reconstituted nucleosomes in vitro.

网站特定赖氨酸乙酰化的组蛋白为核体重组使用遗传密码扩展在 埃斯切里西亚大肠杆菌

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

生物化学

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

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

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

用质谱和荧光法研究非规范氨基酸和蛋白质分析的选择性压力下工程 "金" 荧光

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

生物工程

Antimicrobial Peptides Produced by Selective Pressure Incorporation of Non-canonical Amino Acids

Nature has a variety of possibilities to create new protein functions by modifying the sequence of the individual amino acid building blocks. However, all variations are based on the 20 canonical amino acids (cAAs). As a way to introduce additional physicochemical properties into polypeptides, the incorporation of non-canonical amino acids (ncAAs) is increasingly used in protein engineering. Due to their relatively short length, the modification of ribosomally synthesized and post-translationally modified peptides by ncAAs is particularly attractive. New functionalities and chemical handles can be generated by specific modifications of individual residues. The selective pressure incorporation (SPI) method utilizes auxotrophic host strains that are deprived of an essential amino acid in chemically defined growth media. Several structurally and chemically similar amino acid analogs can then be activated by the corresponding aminoacyl-tRNA synthetase and provide residue-specific cAA(s) &#8594; ncAA(s) substitutions in the target peptide or protein sequence. Although, in the context of the SPI method, ncAAs are also incorporated into the host proteome during the phase of recombinant gene expression, the majority of the cell&#39;s resources are assigned to the expression of the target gene. This enables efficient residue-specific incorporation of ncAAs often accompanied with high amounts of modified target. The presented work describes the in vivo incorporation of six proline analogs into the antimicrobial peptide nisin, a lantibiotic naturally produced by Lactococcus lactis. Antimicrobial properties of nisin can be changed and further expanded during its fermentation and expression in auxotrophic Escherichia coli strains in defined growth media. Thereby, the effects of residue-specific replacement of cAAs with ncAAs can deliver changes in antimicrobial activity and specificity. Antimicrobial activity assays and fluorescence microscopy are used to test the new nisin variants for growth inhibition of a Gram-positive Lactococcus lactis indicator strain. Mass spectroscopy is used to confirm ncAA incorporation in bioactive nisin variants.

The protocol presents the Escherichia coli-based selective pressure incorporation of non-canonical amino acids (ncAAs) into the lactococcal antimicrobial peptide nisin. Its properties can be changed during recombinant expression via substitution with desired ncAAs in defined growth media. Resulting changes in bioactivity are mapped by growth inhibition assays and fluorescence microscopy.

非规范氨基酸选择性压力合成抗菌肽的研究

Nature has a variety of possibilities to create new protein functions by modifying the sequence of the individual amino acid building blocks. However, all ...

Targeting Cysteine Thiols for in Vitro Site-specific Glycosylation of Recombinant Proteins

Stromal interaction molecule-1 (STIM1) is a type-I transmembrane protein located on the endoplasmic reticulum (ER) and plasma membranes (PM). ER-resident STIM1 regulates the activity of PM Orai1 channels in a process known as store operated calcium (Ca2+) entry which is the principal Ca2+ signaling process that drives the immune response. STIM1 undergoes post-translational N-glycosylation at two luminal Asn sites within the Ca2+ sensing domain of the molecule. However, the biochemical, biophysical, and structure biological effects of N-glycosylated STIM1 were poorly understood until recently due to an inability to readily obtain high levels of homogeneous N-glycosylated protein. Here, we describe the implementation of an in vitro chemical approach which attaches glucose moieties to specific protein sites applicable to understanding the underlying effects of N-glycosylation on protein structure and mechanism. Using solution nuclear magnetic resonance spectroscopy we assess both efficiency of the modification as well as the structural consequences of the glucose attachment with a single sample. This approach can readily be adapted to study the myriad glycosylated proteins found in nature.

Targeting Cysteine Thiols for in Vitro Site-specific Glycosylation of Recombinant Proteins

Biochemical and structural analyses of glycosylated proteins require relatively large amounts of homogeneous samples. Here, we present an efficient chemical method for site-specific glycosylation of recombinant proteins purified from bacteria by targeting reactive Cys thiols.

靶向半胱氨酸硫用于体外重组蛋白的特异糖基化

Stromal interaction molecule-1 (STIM1) is a type-I transmembrane protein located on the endoplasmic reticulum (ER) and plasma membranes (PM). ER-resident ...

免疫与感染

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

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

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

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

在大肠杆菌中生成特异的乙酰蛋白的简易协议

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

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

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

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

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

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

Cell-Type Specific Protein Purification and Identification from Complex Tissues Using a Mutant Methionine tRNA Synthetase Mouse Line

Understanding protein homeostasis in vivo is key to knowing how the cells work in both physiological and disease conditions. The present protocol describes in vivo labeling and subsequent purification of newly synthesized proteins using an engineered mouse line to direct protein labeling to specific cellular populations. It is an inducible line by Cre recombinase expression of L274G-Methionine tRNA synthetase (MetRS*), enabling azidonorleucine (ANL) incorporation to the proteins, which otherwise will not occur. Using the method described here, it is possible to purify cell-type-specific proteomes labeled in vivo and detect subtle changes in protein content due to sample complexity reduction.

This protocol describes how to perform cell-type-specific protein labeling with azidonorleucine (ANL) using a mouse line expressing a mutant L274G-Methionine tRNA synthetase (MetRS*) and the necessary steps for labeled cell-type-specific proteins isolation. We outline two possible ANL administration routes in live mice by (1) drinking water and (2) intraperitoneal injections.

使用突变蛋氨酸tRNA合成酶小鼠系从复杂组织中纯化和鉴定细胞类型特异性蛋白

Understanding protein homeostasis in vivo is key to knowing how the cells work in both physiological and disease conditions. The present protocol ...

神经科学

CAPRRESI: Chimera Assembly by Plasmid Recovery and Restriction Enzyme Site Insertion

Here, we present chimera assembly by plasmid recovery and restriction enzyme site insertion (CAPRRESI). CAPRRESI benefits from many strengths of the original plasmid recovery method and introduces restriction enzyme digestion to ease DNA ligation reactions (required for chimera assembly). For this protocol, users clone wildtype genes into the same plasmid (pUC18 or pUC19). After the in silico selection of amino acid sequence regions where chimeras should be assembled, users obtain all the synonym DNA sequences that encode them. Ad hoc Perl scripts enable users to determine all synonym DNA sequences. After this step, another Perl script searches for restriction enzyme sites on all synonym DNA sequences. This in silico analysis is also performed using the ampicillin resistance gene (ampR) found on pUC18/19 plasmids. Users design oligonucleotides inside synonym regions to disrupt wildtype and ampR genes by PCR. After obtaining and purifying complementary DNA fragments, restriction enzyme digestion is accomplished. Chimera assembly is achieved by ligating appropriate complementary DNA fragments. pUC18/19 vectors are selected for CAPRRESI because they offer technical advantages, such as small size (2,686 base pairs), high copy number, advantageous sequencing reaction features, and commercial availability. The usage of restriction enzymes for chimera assembly eliminates the need for DNA polymerases yielding blunt-ended products. CAPRRESI is a fast and low-cost method for fusing protein-coding genes.

Here, we present chimera assembly by plasmid recovery and restriction enzyme site insertion (CAPRRESI), a protocol based on the insertion of restriction enzyme sites into synonym DNA sequences and functional plasmid recovery. This protocol is a fast and low-cost method for fusing protein-coding genes.

CAPRRESI:通过质粒恢复和限制酶位点插入的嵌合体装配

Here, we present chimera assembly by plasmid recovery and restriction enzyme site insertion (CAPRRESI). CAPRRESI benefits from many strengths of the ...

非经典氨基酸的具体残留纳入到模型蛋白质使用

摘要

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此视频中的章节

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

大肠杆菌无细胞蛋白合成: 一种强大、灵活和可访问的平台技术的协议

网站特定赖氨酸乙酰化的组蛋白为核体重组使用遗传密码扩展在埃斯切里西亚大肠杆菌

用质谱和荧光法研究非规范氨基酸和蛋白质分析的选择性压力下工程 "金" 荧光

非规范氨基酸选择性压力合成抗菌肽的研究

靶向半胱氨酸硫用于体外重组蛋白的特异糖基化

在大肠杆菌中生成特异的乙酰蛋白的简易协议

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

使用突变蛋氨酸tRNA合成酶小鼠系从复杂组织中纯化和鉴定细胞类型特异性蛋白

CAPRRESI:通过质粒恢复和限制酶位点插入的嵌合体装配

非经典氨基酸的具体残留纳入到模型蛋白质使用

摘要

探索更多视频

此视频中的章节

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

大肠杆菌无细胞蛋白合成: 一种强大、灵活和可访问的平台技术的协议

网站特定赖氨酸乙酰化的组蛋白为核体重组使用遗传密码扩展在 埃斯切里西亚大肠杆菌

用质谱和荧光法研究非规范氨基酸和蛋白质分析的选择性压力下工程 "金" 荧光

非规范氨基酸选择性压力合成抗菌肽的研究

靶向半胱氨酸硫用于体外重组蛋白的特异糖基化

在大肠杆菌中生成特异的乙酰蛋白的简易协议

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

使用突变蛋氨酸tRNA合成酶小鼠系从复杂组织中纯化和鉴定细胞类型特异性蛋白

CAPRRESI:通过质粒恢复和限制酶位点插入的嵌合体装配

网站特定赖氨酸乙酰化的组蛋白为核体重组使用遗传密码扩展在埃斯切里西亚大肠杆菌