这项工作由以色列科学基金会结构细胞生物学研究中心（I-CORE）（1775/12）和以色列科学基金会资助，授予1721/16和2338/16（YH）和825/14（DK ）。对"田园遗产基金会"的支持表示高度赞赏。这项工作由Ilan Edri和Michal Goldenberg完成，部分履行了特拉维夫大学萨克勒医学院的MD论文要求。

1。克隆顺式 -PT在大肠杆菌中过表达 <ol><li>获得pET-32b表达载体，设计用于与109aa硫氧还蛋白（TRX）蛋白17融合的蛋白质序列的克隆和高水平表达，以及全长顺式 -PT的大肠杆菌密码子优化的18编码序列。 </li><li>照顾到具有TEV蛋白酶（烟草蚀纹病毒蛋白酶）切割位点（ENLYFQ / G，其中，"/"表示切割点）9，随后通过短的柔性接头（SGSGSG，以增强切割位点可访问）的顺式的上游-PT序列（ 图 1A ）。另外，添加合适的5&#39;和3&#39;限制性位点进行克隆。 </li><li>克隆合成的DNA构建体导入利用标准连接技术10所述的pET-32b的载体。 </li></ol> 2。 Overexpre人DHDDS在大肠杆菌中的表达 <ol><li>转化大肠杆菌 T7根据制造商的说明书用TRX-融合DHDDS构建体表达感受态细胞。 </li><li>在氨苄青霉素选择条件（200 mg / L）下，按照制造商的说明在LB-琼脂上培养转化的细胞。 </li><li>将选择的菌落接种到250mL烧瓶中的100mL LB培养基中，培养基用100mg / L氨苄青霉素，并在37℃下恒温摇动200rpm培养过夜。 </li><li>将80mL培养物接种到两个5L烧瓶（每个烧瓶40mL）中，每个含有1.5L 2x YT培养基（16g / L胰蛋白胨，10g / L酵母提取物，5g / L NaCl），100mg / L氨苄青霉素。 </li><li>孵育培养物在37℃，以180rpm恒定振荡直到达到OD 600nm 处 = 0.5。 </li><li>将温度降至16℃，加入0.5mM异丙基β-D-1-硫代吡喃半乳糖苷（IPTG）诱导细胞。精读在相同条件下孵育16-20小时进行蛋白质表达。 </li><li>通过离心（10,000× g ，10分钟）收获细胞。 注意:协议可以暂停在这里;细胞沉淀物应保持在-80℃直至纯化。 </li></ol> 3。人类DHDDS的纯化<ol><li>将细胞重悬于缓冲液A（25mM Tris-HCl，pH7.5,150mM NaCl，0.1％Triton X-100,10mMβ-巯基乙醇），1μg/ mL DNA酶I和蛋白酶抑制剂混合物中。 </li><li>使用玻璃 - 特氟龙均质器均化细胞。 </li><li>使用微流化器或相当于12,000 - 15,000 psi 11的电池破坏电池。 </li><li>在4℃下以40,000xg离心细胞裂解物45分钟。回收含有可溶性级分的上清液。 </li><li>将上清液加载到10 - 10 mL固定化钴固定化金属亲和层析（Co 2+ -IMAC） 12柱上mM咪唑，然后用缓冲液A和10mM咪唑充分洗涤以减少非特异性蛋白质结合。 </li><li>用补充有250mM咪唑的缓冲液A洗脱过表达的蛋白质。 </li><li>用缓冲液A平衡的53 mL制备型脱盐柱除去咪唑。 </li><li>将6xHis标记的TEV蛋白酶（每50mg蛋白质1mg TEV蛋白酶）加入洗脱的蛋白质中以在4℃下将其6xHis标记的TRX融合蛋白去除过夜。 </li><li>将切割的蛋白质加载到Co 2+ -IMAC柱上，用补充有5mM咪唑的缓冲液A平衡以除去切割的6x His标记的TRX融合蛋白和TEV蛋白酶。 </li><li>收集流通并浓缩至3-4 mL，使用30 kDa分子量截留离心过滤器。 </li><li>将浓缩的蛋白质加载到用缓冲液A平衡的superdex-200大小排阻色谱柱上，以进行最终纯化。蛋白质作为二聚体（〜76 kDa）洗脱。 </LI&gt; <li>通过比较洗脱曲线与色谱柱校准曲线来选择相关级分。 </li><li>使用SDS-PAGE评估制剂的纯度。 </li><li>确定下游实验所需的蛋白质浓度和纯化的浓缩蛋白质在液氮中的闪蒸等分试样，并储存在-80°C直到使用。 </li></ol> 4。分析尺寸排阻色谱法（SEC） <ol><li>为了确保蛋白质耐受冻融循环的能力，解冻冷冻蛋白质的一部分，并以21,000 x g离心10分钟以除去不溶性聚集体。收集上清液。 </li><li>使用超高效液相色谱系统13将蛋白质加载到用25mM Tris-HCl，pH 7.5,150mM NaCl，0.02％Triton X-100,10mMβ-巯基乙醇预平衡的superdex-200分析柱上， 14 。 </li><li>监测色氨酸荧光scance（ λex = 280nm， λem = 350nm）以检测蛋白质的洗脱洗脱曲线。确保具有有效的分子量评估校准曲线 - 这些曲线可通过 SEC色谱柱制造商获得。 </li></ol> 5。酶动力学-与时间相关的活动15，16，17 <ol><li>为了确保纯化的蛋白质的活性，混合纯化的DHDDS的5μM的10μMFPP和50μM14 C-IPP以引发在缓冲液A中的反应用0.5mM的MgCl 2，在22 - 30℃。 注意:精确的反应条件可能随着不同于DHDDS的顺式 -PT的制备而变化。此外，DHDDS的活性也取决于温度和[Mg 2+ ]。 注意: 14 C-IPP是放射性的，应根据当地的辐射安全规定使用。 </LI&gt; <li>通过加入补充20mM EDTA的15μL缓冲液A（终浓度为10mM EDTA）立即骤冷反应，在0,2,4和6小时从反应中取出15μL样品。 </li><li>加入1mL H 2 O饱和的1-丁醇并充分涡旋以提取反应产物。 注意:可以在此处停止协议，并保留样品供以后读取。 </li><li>向样品中加入闪烁混合物，并使用闪烁计数器，定量包含14 C的丁醇相中的反应产物，以及反应混合物的放射性为15μL，代表总放射性。 </li><li>减去从每个时间点使用0小时样本测量的背景读数，并计算使用的14 C-IPP的百分比。 </li><li>在每个时间点计算14 C-IPP净值，计算从总共14个使用的百分比</sup&gt; C-IPP浓度，并将结果绘制为时间的函数。预期净14 C-IPP合并增加。 </li></ol>

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作者没有什么可以披露的。

这里描述的用于纯化大肠杆菌细胞中功能性人DHDDS的方案是简单和有效的，允许一旦合适的构建体可用，在3-4天内过表达和纯化蛋白质。鉴于基因组测序的突破，蛋白质纯化的这些方案具有特殊的意义，这提供了许多关于许多疾病18遗传学的信息，因此需要开发高通量方法来表征蛋白质级19的致病机制。 为了克服异源蛋白过表达和纯?...

异戊二烯基转移是一组催化经由多个缩合反应1使用异戊烯二磷酸（IPP）2烯丙基二磷酸，的链延长酶。 Z型酶催化从缩合反应形成顺式双键，而E型酶催化反式双键形成3 。 顺式 -磷酸转移酶（ 顺式 -PT，Z型酶）根据其产物链长度经常分类为短链（C 15 ），中链（C 50-55 ）和长链（C 70-120 ） 4 。 DHDDS（脱氢二酰基二磷酸合成酶）是一种真核长链顺式 -PT，它通过与异戊烯基二磷酸（IPP） 1的多次缩合催化法呢基二磷酸（FPP，烯丙基二磷酸）的链延长，5，6。这导致脱氢二酰基二磷酸盐的形成，C 55-100多聚丙烯酰二磷酸酯作为焦磷酸二酰基焦磷酸酯的前体，参与N-连接蛋白糖基化的糖基载体分子1 。其中德系犹太人，在DHDDS导致常染色体隐性色素性视网膜炎7,8的错义非保守突变（K42E）。因此，本方案是为了获得适用于机械研究的纯化DHDDS而开发的。 大肠杆菌被认为是重要蛋白质表达最方便和最具成本效益的宿主，因此也是最常用的宿主。然而，当试图在大肠杆菌中异源过表达蛋白质时，应该考虑蛋白质特异性的考虑。获得正确的折叠，活动来自大肠杆菌的重组蛋白质不是由于不同蛋白质的不同特性而简单的。已经开发了许多方法来克服这些障碍。在这里，蛋白质融合的使用，优化的培养条件和密码子优化被用于允许功能活跃的人DHDDS在大肠杆菌中的过表达和纯化。值得注意的是，以前尝试过度表达酵母顺式 -PT而没有蛋白质融合，因为即使在洗涤剂12的存在下也完全不溶，因此不成功。所述方案简单，经济有效，节省时间，并允许获得适合机械研究的DHDDS制剂。鉴于不同物种中顺式 -PT的同源性，我们建议该方案也可以应用于其他真核的顺式 -PT。

<table><tbody><tr><td>pET-32b</td><td>Novagen</td><td>69016-3</td><td></td></tr><tr><td>T7 Express&amp;nbsp;lysY&amp;nbsp;Competent&amp;nbsp;&lt;em&gt;E. coli&lt;/em&gt;&amp;nbsp;(High Efficiency)</td><td>NEB</td><td>C3010I</td><td></td></tr><tr><td>cOmplete, EDTA-free Protease Inhibitor Cocktail</td><td>Roche</td><td>11873580001</td><td></td></tr><tr><td>TALON-superflow resin</td><td>GE Healthcare</td><td>28-9574-99</td><td></td></tr><tr><td>HiPrep 26/10 desalting column&amp;nbsp;</td><td>GE Healthcare</td><td>17508701</td><td></td></tr><tr><td>HiLoad 16/60 superdex-200&amp;nbsp;</td><td>GE Healthcare</td><td>28989335</td><td></td></tr><tr><td>superdex-200 increase 5/150 GL&amp;nbsp;</td><td>GE Healthcare</td><td>28990945</td><td></td></tr><tr><td>&lt;sup&gt;14&lt;/sup&gt;C-Isopentenyl pyrophosphate</td><td>Perkin-Elmer</td><td>NEC773050UC&amp;nbsp;</td><td></td></tr><tr><td>trans,trans-Farnesyl pyrophosphate</td><td>Sigma</td><td>44270-10MG</td><td></td></tr></tbody></table>

overexpression and purification of human cis-prenyltransferase in escherichia coli

丙二酰转移酶（PT）是一组通过多次缩合反应使用异戊烯基二磷酸酯（IPP）催化烯丙基二磷酸的链延长的酶。 DHDDS（脱氢二酰基二磷酸合成酶）是通过与异戊烯基二磷酸酯（IPP）的多次缩合催化法呢二磷酸（FPP，一种烯丙基二磷酸酯）催化链延长的真核长链顺式 -PT（从缩合反应形成顺式双键）。 DHDDS具有生物医学重要性，因为酶中的非保守突变（K42E）导致色素性视网膜炎 ，最终导致失明。因此，本协议是为了获得大量纯化的DHDDS而开发的，适用于机械研究。在这里，蛋白质融合的使用，优化的培养条件和密码子优化被用于允许功能活跃的人类DHDDS的过表达和纯化<e米&gt;电子。大肠杆菌描述的协议是简单，成本效益和时间节省。不同物种中顺式 -PT的同源性表明，该方案也可以应用于其他真核的顺式 -PT，如涉及天然橡胶合成的那些。

这里使用的构造和纯化过程的一般概述如图1所示。在每个纯化步骤获得的样品如图2所示。该SDS-PAGE分析显示DHDDS的逐步纯化，得到高度纯化的产物。 图3显示了纯化酶的分析SEC的结果，显示蛋白质仅被观察为同二聚体。 图4显示代表性的时间依赖性活性测定。 14 C-IPP掺入明显?...

Watch this Scientific Journal Video about Overexpression and Purification of Human Cis-prenyltransferase in Escherichia coli at JoVE.com

Overexpression and Purification of Human Cis-prenyltransferase in Escherichia coli

来自大肠杆菌的非变性条件下用于过表达和纯化密码子优化的人顺式 -丙烯基转移酶的简单方案 大肠杆菌 ，以及酶活性测定法。该方案可以推广用于生产适合于机械研究的数量和质量的其它顺式异戊烯基转移酶蛋白质。

人类的过表达和纯化<em&gt;顺</emβ-肾上腺素转移酶<em&gt;大肠杆菌</em

Prenyltransferases (PT) are a group of enzymes that catalyze chain elongation of allylic diphosphate using isopentenyl diphosphate (IPP) via multiple ...

overexpression-purification-human-cis-prenyltransferase-escherichia

Universidad de Cartagena UDEC

Research

JoVE Journal

Immunology and Infection

Overexpression and Purification of Human Cis-prenyltransferase in Escherichia coli

6.6K 次观看.  Tel Aviv University. 来自大肠杆菌的非变性条件下用于过表达和纯化密码子优化的人顺式 -丙烯基转移酶的简单方案 大肠杆菌 ，以及酶活性测定法。该方案可以推广用于生产适合于机械研究的数量和质量的其它顺式异戊烯基转移酶蛋白质。 

视频: Overexpression and Purification of Human Cis-prenyltransferase in Escherichia coli

Inducible T7 RNA Polymerase-mediated Multigene Expression System, pMGX

Co-expression of multiple proteins is increasingly essential for synthetic biology, studying protein-protein complexes, and characterizing and harnessing biosynthetic pathways. In this manuscript, the use of a highly effective system for the construction of multigene synthetic operons under the control of an inducible T7 RNA polymerase is described. This system allows many genes to be expressed simultaneously from one plasmid. Here, a set of four related vectors, pMGX-A, pMGX-hisA, pMGX-K, and pMGX-hisK, with either the ampicillin or kanamycin resistance selectable marker (A and K) and either possessing or lacking an N-terminal hexahistidine tag (his) are disclosed. Detailed protocols for the construction of synthetic operons using this vector system are provided along with the corresponding data, showing that a pMGX-based system containing five genes can be readily constructed and used to produce all five encoded proteins in Escherichia coli. This system and protocol enables researchers to routinely express complex multi-component modules and pathways in E. coli.

This study describes methods for the T7-mediated co-expression of multiple genes from a single plasmid in Escherichia coli using the pMGX plasmid system.

可诱导T7 RNA聚合酶介导的多基因表达系统，pMGX

Co-expression of multiple proteins is increasingly essential for synthetic biology, studying protein-protein complexes, and characterizing and harnessing ...

科研

遗传学

High Precision FRET at Single-molecule Level for Biomolecule Structure Determination

A protocol on how to perform high-precision interdye distance measurements using F&#246;rster resonance energy transfer (FRET) at the single-molecule level in multiparameter fluorescence detection (MFD) mode is presented here. MFD maximizes the usage of all &#34;dimensions&#34; of fluorescence to reduce photophysical and experimental artifacts and allows for the measurement of interdye distance with an accuracy up to ~1 &#197; in rigid biomolecules. This method was used to identify three conformational states of the ligand-binding domain of the N-methyl-D-aspartate (NMDA) receptor to explain the activation of the receptor upon ligand binding. When comparing the known crystallographic structures with experimental measurements, they agreed within less than 3 &#197; for more dynamic biomolecules. Gathering a set of distance restraints that covers the entire dimensionality of the biomolecules would make it possible to provide a structural model of dynamic biomolecules.

A protocol for high-precision FRET experiments at the single molecule level is presented here. Additionally, this methodology can be used to identify three conformational states in the ligand-binding domain of the N-methyl-D-aspartate (NMDA) receptor. Determining precise distances is the first step towards building structural models based on FRET experiments.

单分子水平的高精度FRET用于生物分子结构测定

A protocol on how to perform high-precision interdye distance measurements using F&#246;rster resonance energy transfer (FRET) at the single-molecule ...

生物化学

Efficient Purification and LC-MS/MS-based Assay Development for Ten-Eleven Translocation-2 5-Methylcytosine Dioxygenase

The epigenetic transcription regulation mediated by 5-methylcytosine (5mC) has played a critical role in eukaryotic development. Demethylation of these epigenetic marks is accomplished by sequential oxidation by ten-eleven translocation dioxygenases (TET1-3), followed by the thymine-DNA glycosylase-dependent base excision repair. Inactivation of the TET2 gene due to genetic mutations or by other epigenetic mechanisms is associated with a poor prognosis in patients with diverse cancers, especially hematopoietic malignancies. Here, we describe an efficient single step purification of enzymatically active untagged human TET2 dioxygenase using cation exchange chromatography. We further provide a liquid chromatography-tandem mass spectrometry (LC-MS/MS) approach that can separate and quantify the four normal DNA bases (A, T, G, and C), as well as the four modified cytosine bases (5-methyl, 5-hydroxymethyl, 5-formyl, and 5-carboxyl). This assay can be used to evaluate the activity of wild type and mutant TET2 dioxygenases.

Here, we present a protocol for an efficient single step purification of the active untagged human ten-eleven translocation-2 (TET2) 5-methylcytosine dioxygenase using ion-exchange chromatography and its assay using a liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based approach.

高效纯化和基于 LC ms/ms 的十-十一 Translocation-2 5-甲基胞嘧啶酶的检测开发

The epigenetic transcription regulation mediated by 5-methylcytosine (5mC) has played a critical role in eukaryotic development. Demethylation of these ...

Expression, Isolation, and Purification of Soluble and Insoluble Biotinylated Proteins for Nerve Tissue Regeneration

Recombinant protein engineering has utilized Escherichia coli (E. coli) expression systems for nearly 4 decades, and today E. coli is still the most widely used host organism. The flexibility of the system allows for the addition of moieties such as a biotin tag (for streptavidin interactions) and larger functional proteins like green fluorescent protein or cherry red protein. Also, the integration of unnatural amino acids like metal ion chelators, uniquely reactive functional groups, spectroscopic probes, and molecules imparting post-translational modifications has enabled better manipulation of protein properties and functionalities. As a result this technique creates customizable fusion proteins that offer significant utility for various fields of research. More specifically, the biotinylatable protein sequence has been incorporated into many target proteins because of the high affinity interaction between biotin with avidin and streptavidin. This addition has aided in enhancing detection and purification of tagged proteins as well as opening the way for secondary applications such as cell sorting. Thus, biotin-labeled molecules show an increasing and widespread influence in bioindustrial and biomedical fields. For the purpose of our research we have engineered recombinant biotinylated fusion proteins containing nerve growth factor (NGF) and semaphorin3A (Sema3A) functional regions. We have reported previously how these biotinylated fusion proteins, along with other active protein sequences, can be tethered to biomaterials for tissue engineering and regenerative purposes. This protocol outlines the basics of engineering biotinylatable proteins at the milligram scale, utilizing&#160; a T7 lac inducible vector and E. coli expression hosts, starting from transformation to scale-up and purification.

Developing biotinylatable fusion proteins has many potential applications in various fields of research. Recombinant protein engineering is a straight forward procedure that is cost-effective, providing high yields of custom-designed proteins.

表达，分离，纯化可溶性和不溶性蛋白质生物素对神经组织再生

Recombinant protein engineering has utilized Escherichia coli (E. coli) expression systems for nearly 4 decades, and today E. coli is still the most ...

生物工程

A Customizable Approach for the Enzymatic Production and Purification of Diterpenoid Natural Products

Diterpenoids form a diverse class of small molecule natural products that are widely distributed across the kingdoms of life and have critical biological functions in developmental processes, interorganismal interactions, and environmental adaptation. Due to these various bioactivities, many diterpenoids are also of economic importance as pharmaceuticals, food additives, biofuels, and other bioproducts. Advanced genomics and biochemical approaches have enabled a rapid increase in the knowledge of diterpenoid-metabolic genes, enzymes, and pathways. However, the structural complexity of diterpenoids and the narrow taxonomic distribution of individual compounds in often only a single species remain constraining factors for their efficient production. Availability of a broader range of metabolic enzymes now provide resources for producing diterpenoids in sufficient titers and purity to facilitate a deeper investigation of this important metabolite group. Drawing on established tools for microbial and plant-based enzyme co-expression, we present an easily operated and customizable protocol for the enzymatic production of diterpenoids in either Escherichia coli or Nicotiana benthamiana, and the purification of desired products via silica chromatography and semi-preparative HPLC. Using the group of maize (Zea mays) dolabralexin diterpenoids as an example, we highlight how modular combinations of diterpene synthase (diTPS) and cytochrome P450 monooxygenase (P450) enzymes can be used to generate different diterpenoid scaffolds. Purified compounds can be used in various downstream applications, such as metabolite structural analyses, enzyme structure-function studies, and in vitro and in planta bioactivity experiments.

Here we present easy to use protocols for producing and purifying diterpenoid metabolites through the combinatorial expression of biosynthetic enzymes in Escherichia coli or Nicotiana benthamiana, followed by chromatographic product purification. The resulting metabolites are suitable for various studies including molecular structure characterization, enzyme functional studies, and bioactivity assays.

一种可定制的酶化生产与纯化天然产物的方法

Diterpenoids form a diverse class of small molecule natural products that are widely distributed across the kingdoms of life and have critical biological ...

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 ...

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 Convenient and General Expression Platform for the Production of Secreted Proteins from Human Cells

Recombinant protein expression in bacteria, typically E. coli, has been the most successful strategy for milligram quantity expression of proteins. However, prokaryotic hosts are often not as appropriate for expression of human, viral or eukaryotic proteins due to toxicity of the foreign macromolecule, differences in the protein folding machinery, or due to the lack of particular co- or post-translational modifications in bacteria. Expression systems based on yeast (P. pastoris or S. cerevisiae) 1,2, baculovirus-infected insect (S. frugiperda or T. ni) cells 3, and cell-free in vitro translation systems 2,4 have been successfully used to produce mammalian proteins. Intuitively, the best match is to use a mammalian host to ensure the production of recombinant proteins that contain the proper post-translational modifications. A number of mammalian cell lines (Human Embryonic Kidney (HEK) 293, CV-1 cells in Origin carrying the SV40 larget T-antigen (COS), Chinese Hamster Ovary (CHO), and others) have been successfully utilized to overexpress milligram quantities of a number of human proteins 5-9. However, the advantages of using mammalian cells are often countered by higher costs, requirement of specialized laboratory equipment, lower protein yields, and lengthy times to develop stable expression cell lines. Increasing yield and producing proteins faster, while keeping costs low, are major factors for many academic and commercial laboratories.
Here, we describe a time- and cost-efficient, two-part procedure for the expression of secreted human proteins from adherent HEK 293T cells. This system is capable of producing microgram to milligram quantities of functional protein for structural, biophysical and biochemical studies. The first part, multiple constructs of the gene of interest are produced in parallel and transiently transfected into adherent HEK 293T cells in small scale. The detection and analysis of recombinant protein secreted into the cell culture medium is performed by western blot analysis using commercially available antibodies directed against a vector-encoded protein purification tag. Subsequently, suitable constructs for large-scale protein production are transiently transfected using polyethyleneimine (PEI) in 10-layer cell factories. Proteins secreted into litre-volumes of conditioned medium are concentrated into manageable amounts using tangential flow filtration, followed by purification by anti-HA affinity chromatography. The utility of this platform is proven by its ability to express milligram quantities of cytokines, cytokine receptors, cell surface receptors, intrinsic restriction factors, and viral glycoproteins. This method was also successfully used in the structural determination of the trimeric ebolavirus glycoprotein 5,10.
In conclusion, this platform offers ease of use, speed and scalability while maximizing protein quality and functionality. Moreover, no additional equipment, other than a standard humidified CO2 incubator, is required. This procedure may be rapidly expanded to systems of greater complexity, such as co-expression of protein complexes, antigens and antibodies, production of virus-like particles for vaccines, or production of adenoviruses or lentiviruses for transduction of difficult cell lines.

In the post-human genomics era, the availability of recombinant proteins in native conformations is crucial to structural, functional and therapeutic research and development. Here, we describe a test- and large-scale protein expression system in human embryonic kidney 293T cells that can be used to produce a variety of recombinant proteins.

一个从人体细胞分泌蛋白生产的方便，一般的表达平台

Recombinant protein expression in bacteria, typically E. coli, has been the most successful strategy for milligram quantity expression of proteins. ...

生物学

Protocols for Implementing an Escherichia coli Based TX-TL Cell-Free Expression System for Synthetic Biology

Ideal cell-free expression systems can theoretically emulate an in vivo cellular environment in a controlled in vitro platform.1 This is useful for expressing proteins and genetic circuits in a controlled manner as well as for providing a prototyping environment for synthetic biology.2,3 To achieve the latter goal, cell-free expression systems that preserve endogenous Escherichia coli transcription-translation mechanisms are able to more accurately reflect in vivo cellular dynamics than those based on T7 RNA polymerase transcription. We describe the preparation and execution of an efficient endogenous E. coli based transcription-translation (TX-TL) cell-free expression system that can produce equivalent amounts of protein as T7-based systems at a 98% cost reduction to similar commercial systems.4,5 The preparation of buffers and crude cell extract are described, as well as the execution of a three tube TX-TL reaction. The entire protocol takes five days to prepare and yields enough material for up to 3000 single reactions in one preparation. Once prepared, each reaction takes under 8 hr from setup to data collection and analysis. Mechanisms of regulation and transcription exogenous to E. coli, such as lac/tet repressors and T7 RNA polymerase, can be supplemented.6 Endogenous properties, such as mRNA and DNA degradation rates, can also be adjusted.7 The TX-TL cell-free expression system has been demonstrated for large-scale circuit assembly, exploring biological phenomena, and expression of proteins under both T7- and endogenous promoters.6,8 Accompanying mathematical models are available.9,10 The resulting system has unique applications in synthetic biology as a prototyping environment, or "TX-TL biomolecular breadboard."

Protocols for Implementing an Escherichia coli Based TX-TL Cell-Free Expression System for Synthetic Biology

This five-day protocol outlines all steps, equipment, and supplemental software necessary for creating and running an efficient endogenous Escherichia coli based TX-TL cell-free expression system from scratch. With reagents, the protocol takes 8 hours or less to setup a reaction, collect, and process data.

协议用于实现<em&gt;大肠杆菌</em&gt;基于TX-TL无细胞表达系统合成生物学

Ideal cell-free expression systems can theoretically emulate an in vivo cellular environment in a controlled in vitro platform.1 This is useful for ...

Direct Protein Delivery to Mammalian Cells Using Cell-permeable Cys2-His2 Zinc-finger Domains

Due to their modularity and ability to be reprogrammed to recognize a wide range of DNA sequences, Cys2-His2 zinc-finger DNA-binding domains have emerged as useful tools for targeted genome engineering. Like many other DNA-binding proteins, zinc-fingers also possess the innate ability to cross cell membranes. We recently demonstrated that this intrinsic cell-permeability could be leveraged for intracellular protein delivery. Genetic fusion of zinc-finger motifs leads to efficient transport of protein and enzyme cargo into a broad range of mammalian cell types. Unlike other protein transduction technologies, delivery via zinc-finger domains does not inhibit enzyme activity and leads to high levels of cytosolic delivery. Here a detailed step-by-step protocol is presented for the implementation of zinc-finger technology for protein delivery into mammalian cells. Key steps for achieving high levels of intracellular zinc-finger-mediated delivery are highlighted and strategies for maximizing the performance of this system are discussed.

Direct Protein Delivery to Mammalian Cells Using Cell-permeable Cys2-His2 Zinc-finger Domains

Zinc-finger domains are intrinsically cell-permeable and capable of mediating protein delivery into a broad range of mammalian cell types. Here, a detailed step-by-step protocol for implementing zinc-finger technology for intracellular protein delivery is presented.

蛋白质直接送货到哺乳动物细胞利用细胞渗透半胱氨酸<sub&gt; 2</sub&gt; -His<sub&gt; 2</sub&gt;锌指域

Due to their modularity and ability to be reprogrammed to recognize a wide range of DNA sequences, Cys2-His2 zinc-finger DNA-binding domains have emerged ...

人类的过表达和纯化

摘要

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