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首页> 外文期刊>Biotechnology Progress >Rapid screening and scale‐up of ultracentrifugation‐free, membrane‐based procedures for purification of His‐tagged membrane proteins
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Rapid screening and scale‐up of ultracentrifugation‐free, membrane‐based procedures for purification of His‐tagged membrane proteins

机译：快速筛选和扩大超速离心，膜的基于膜的方法，用于纯化His标记的膜蛋白

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Abstract > This study develops procedures to rapidly screen conditions for purification of membrane proteins (MPs) using 96‐well plates containing nickel‐functionalized membranes. In addition to their application in the pharmaceutical industry, MPs are important components of new sensors, synthetic membranes, and bioelectronic devices. However, purification of MPs is challenging due to their hydrophobic exterior, which requires stabilization in amphipathic detergent micelles. We examined the extent of extraction of the light‐driven sodium transporter, Krokinobacter eikastus rhodopsin 2 (KR2) heterologously expressed in Escherichia coli using different salts and maltoside‐based detergents. The extraction was followed by subsequent affinity purification in membranes functionalized with Ni 2+ ‐nitrilotriacetate complexes that bind the His‐tagged KR2. We also employed a hydrophobic chelator to separate detergent micelles from the aqueous phase as an initial isolation step prior to affinity purification. Unlike conventional resin‐based capture, which can take a full day or more, the membrane‐based screening of purification conditions takes only a few hours, and its scale‐up involves changing from a 96‐well format to a larger membrane module. The novelty of the method lies in utilizing membrane‐based ultracentrifugation‐free purification of MPs from cell membrane fragments; the optimized purification conditions from the screening method can potentially be applied to large‐scale/conventional resin‐based purification of MPs. </abstract> </span> <span class="z_kbtn z_kbtnclass hoverxs" style="display: none;">展开▼</span> </div> <div class="translation abstracttxt"> <span class="zhankaihshouqi fivelineshidden" id="abstract"> <span>机译：</span><Abstract Type =“Main”XML：Lang =“en”> <标题类型=“main”>抽象</ title> > 该研究使用含有镍官能化膜的96孔板纯化膜蛋白（MPS）的纯化筛选条件的程序。除了在制药业的应用外，MPS还是新传感器，合成膜和生物电子器件的重要组成部分。然而，由于其疏水性外部，MPS的纯化是挑战，这需要在两亲性洗涤剂胶束中稳定化。我们检查了轻推式钠转运蛋白的提取程度， krokinobacter eikastus </ i> rhodopsin 2（KR2）异质地表达 大肠杆菌</ i> 使用不同的盐和基于麦冬的洗涤剂。随后用Ni官能化的膜中的亲和纯化后萃取 2 + </ sup> - 硝基乙酸酯复合物，其结合His标记的KR2。我们还使用疏水性螯合剂，将来自水相的洗涤剂胶束分离为在亲和纯化之前作为初始隔离步骤的初始分离步骤。与常规的基于树脂的捕获不同，这可能需要全天或更长时间，纯化条件的基于膜的筛选只需要几个小时，并且其缩放涉及从96孔格式改变到更大的膜组件。该方法的新颖性在于利用来自细胞膜碎片的MPS基于膜的超四等离子纯化;来自筛选方法的优化纯化条件可能适用于MPS的大规模/常规树脂的净化。 </ p> </摘要> </span> <span class="z_kbtn z_kbtnclass hoverxs" style="display: none;">展开▼</span> </div> </div> <div class="record"> <h2 class="all_title" id="enpatent33" >著录项</h2> <ul> <li> <span class="lefttit">来源</span> <div style="width: 86%;vertical-align: text-top;display: inline-block;"> <a href='/journal-foreign-15005/'>《Biotechnology Progress》</a> <b style="margin: 0 2px;">|</b><span>2019年第6期</span><b style="margin: 0 2px;">|</b><span>共10页</span> </div> </li> <li> <div class="author"> <span class="lefttit">作者</span> <p id="fAuthorthree" class="threelineshidden zhankaihshouqi"> <a href="/search.html?doctypes=4_5_6_1-0_4-0_1_2_3_7_9&sertext=Feroz Hasin&option=202" target="_blank" rel="nofollow">Feroz Hasin;</a> <a href="/search.html?doctypes=4_5_6_1-0_4-0_1_2_3_7_9&sertext=Meisenhelter Joshua&option=202" target="_blank" rel="nofollow">Meisenhelter Joshua;</a> <a href="/search.html?doctypes=4_5_6_1-0_4-0_1_2_3_7_9&sertext=Jokhadze Gia&option=202" target="_blank" rel="nofollow">Jokhadze Gia;</a> <a href="/search.html?doctypes=4_5_6_1-0_4-0_1_2_3_7_9&sertext=Bruening Merlin&option=202" target="_blank" rel="nofollow">Bruening Merlin;</a> <a href="/search.html?doctypes=4_5_6_1-0_4-0_1_2_3_7_9&sertext=Kumar Manish&option=202" target="_blank" rel="nofollow">Kumar Manish;</a> </p> <span class="z_kbtnclass z_kbtnclassall hoverxs" id="zkzz" style="display: none;">展开▼</span> </div> </li> <li> <div style="display: flex;"> <span class="lefttit">作者单位</span> <div style="position: relative;margin-left: 3px;max-width: 639px;"> <div class="threelineshidden zhankaihshouqi" id="fOrgthree"> <p>Department of Chemical EngineeringThe Pennsylvania State UniversityUniversity Park Pennsylvania;</p> <p>Department of Chemical EngineeringThe Pennsylvania State UniversityUniversity Park Pennsylvania;</p> <p>Takara Bio USA Inc.Mountain View California;</p> <p>Department of Chemical and Biomolecular EngineeringUniversity of Notre DameNotre Dame Indiana;</p> <p>Department of Chemical EngineeringThe Pennsylvania State UniversityUniversity Park Pennsylvania;</p> </div> <span class="z_kbtnclass z_kbtnclassall hoverxs" id="zhdw" style="display: none;">展开▼</span> </div> </div> </li> <li > <span class="lefttit">收录信息</span> <span style="width: 86%;vertical-align: text-top;display: inline-block;"></span> </li> <li> <span class="lefttit">原文格式</span> <span>PDF</span> </li> <li> <span class="lefttit">正文语种</span> <span>eng</span> </li> <li> <span class="lefttit">中图分类</span> <span><a href="https://www.zhangqiaokeyan.com/clc/15.html" title="生物科学">生物科学;</a></span> </li> <li class="antistop"> <span class="lefttit">关键词</span> <p style="width: 86%;vertical-align: text-top;"> <a style="color: #3E7FEB;" href="/search.html?doctypes=4_5_6_1-0_4-0_1_2_3_7_9&sertext=affinity purification&option=203" rel="nofollow">affinity purification;</a> <a style="color: #3E7FEB;" href="/search.html?doctypes=4_5_6_1-0_4-0_1_2_3_7_9&sertext=Krokinobacter eikastus rhodopsin 2&option=203" rel="nofollow">Krokinobacter eikastus rhodopsin 2;</a> <a style="color: #3E7FEB;" href="/search.html?doctypes=4_5_6_1-0_4-0_1_2_3_7_9&sertext=membrane adsorber&option=203" rel="nofollow">membrane adsorber;</a> <a style="color: #3E7FEB;" href="/search.html?doctypes=4_5_6_1-0_4-0_1_2_3_7_9&sertext=membrane proteins&option=203" rel="nofollow">membrane proteins;</a> <a style="color: #3E7FEB;" href="/search.html?doctypes=4_5_6_1-0_4-0_1_2_3_7_9&sertext=screening&option=203" rel="nofollow">screening;</a> </p> <div class="translation"> 机译：亲和纯化;krookinobactereikastus rosodopsin 2;膜吸附剂;膜蛋白;筛选; 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target="_blank" rel="nofollow" class="tuijian_auth tuijian_authcolor"> . 刘志敏</a> <a href="/search.html?doctypes=4_5_6_1-0_4-0_1_2_3_7_9&sertext=李哲建&option=202" target="_blank" rel="nofollow" class="tuijian_auth tuijian_authcolor">,李哲建</a> <a href="/search.html?doctypes=4_5_6_1-0_4-0_1_2_3_7_9&sertext=康宁&option=202" target="_blank" rel="nofollow" class="tuijian_auth tuijian_authcolor">,康宁</a> <span> <a href="/journal-cn-57080/" target="_blank" rel="nofollow" class="tuijian_authcolor"> . 分析科学学报 </a> </span> <span> . 2010</span><span>,第1期</span> </span> </div> </li> <li> <div> <b>6. </b><a class="enjiyixqcontent" href="/academic-conference-cn_meeting-19401_thesis/020222211752.html">基于亚甲基蓝一聚合物纳米金抗体多层膜电荷传递的高灵敏免标记免疫检测新方法研究</a> <b>[C]</b> <span> <a href="/search.html?doctypes=4_5_6_1-0_4-0_1_2_3_7_9&sertext=吴再生&option=202" target="_blank" rel="nofollow" class="tuijian_auth tuijian_authcolor"> . 吴再生</a> <a href="/search.html?doctypes=4_5_6_1-0_4-0_1_2_3_7_9&sertext=晨蕊&option=202" target="_blank" rel="nofollow" class="tuijian_auth tuijian_authcolor">,晨蕊</a> <a href="/search.html?doctypes=4_5_6_1-0_4-0_1_2_3_7_9&sertext=松柏&option=202" target="_blank" rel="nofollow" class="tuijian_auth tuijian_authcolor">,松柏</a> <span> <a href="/conference-cn-19401/" target="_blank" rel="nofollow" class="tuijian_authcolor"> . 第十届全国化学传感器学术会议 </a> <span> <span> . 2008</span> </span> </div> </li> <li> <div> <b>7. </b><a class="enjiyixqcontent" href="/academic-degree-domestic_mphd_thesis/020312323091.html">日本血吸虫23KD膜蛋白的原核表达与纯化及双价膜锚定型和分泌型表达疫苗抗感染效果的比较研究</a> <b>[A] </b> <span> <a href="/search.html?doctypes=4_5_6_1-0_4-0_1_2_3_7_9&sertext=邹镇&option=202" target="_blank" rel="nofollow" class="tuijian_auth tuijian_authcolor"> . 邹镇</a> <span> . 2009</span> </span> </div> </li> </ul> <ul style="display: none;"> <li> <div> <b>1. </b><a class="enjiyixqcontent" href="/patent-detail/06120112206042.html">一种含单跨膜区的膜蛋白其跨膜区的纯化方法</a> <b>[P]</b> . <span> 中国专利： CN112210019A </span> <span> . 2021-01-12</span> </div> </li> <li> <div> <b>2. </b><a class="enjiyixqcontent" href="/patent-detail/06120112539633.html">用于纯化核酸探针的纯化膜、纯化柱、纯化试剂盒和纯化方法</a> <b>[P]</b> . <span> 中国专利： CN112553192A </span> <span> . 2021-03-26</span> </div> </li> <li> <div> <b>3. </b><a class="enjiyixqcontent" href="/patent-detail/06130457540739.html">Process for the high-level expression of the outer membrane protein P2 of haemophilus influenzae type B (HIB-P2) in E.coli; purification process of the outer membrane protein P2 or fusion protein thereof, reduplication process of the outer membrane protein P2 or fusion protein obtained; substantially pure reduplicated P2 outer membrane protein from haemophilus influenzae type B (HIB-P2) or fusion protein thereof; vaccine comprising the outer membrane protein P2 of hamophilus influenzae type B (HIB-P2) or a fusion protein thereof; process for obtaining a P2 protein or P2-polysaccharide fusion protein conjugate; process to prevent bacterial meningitis in an animal; pNV-3 vector; pNV-2 vector, and pNV-6 vector</a> <b>[P]</b> . <span> 外国专利： <!-- --> BR9407144A </span> <span> . 1996-09-17</span> </div> <p class="zwjiyix translation" style="max-width: initial;height: auto;word-break: break-all;white-space: initial;text-overflow: initial;overflow: initial;"> <span>机译：乙型流感嗜血杆菌外膜蛋白P2（HIB-P2）在大肠杆菌中高水平表达的方法;外膜蛋白P2或其融合蛋白的纯化过程，外膜蛋白P2或其融合蛋白的重复过程;来自B型流感嗜血杆菌的基本上纯的复制的P2外膜蛋白（HIB-P2）或其融合蛋白;疫苗，其包含B型流感嗜血杆菌的外膜蛋白P2（HIB-P2）或其融合蛋白;获得P2蛋白或P2-多糖融合蛋白缀合物的方法;预防动物细菌性脑膜炎的过程; pNV-3载体; pNV-2矢量和pNV-6矢量 </span> </p> </li> <li> <div> <b>4. </b><a class="enjiyixqcontent" href="/patent-detail/06130457540641.html">Process for high-level expression of the porin group B meningococcal protein or its outer membrane fusion protein in E coli process for the purification of the group B meningococcal porin protein or its external membrane fusion protein meningococcal group B or its fusion protein on the outer membrane porin group B meningococcal protein or its fusion protein on the outer membrane substantially pure vaccine containing</a> <b>[P]</b> . <span> 外国专利： <!-- --> BR9407092A </span> <span> . 1996-09-03</span> </div> <p class="zwjiyix translation" style="max-width: initial;height: auto;word-break: break-all;white-space: initial;text-overflow: initial;overflow: initial;"> <span>机译：在大肠杆菌中高表达B蛋白组B脑膜炎球菌或其外膜融合蛋白的方法在B膜上纯化B组脑膜炎球菌蛋白或其外膜融合蛋白的方法B外膜融合蛋白在外膜上的孔蛋白B组脑膜炎球菌蛋白质或其融合蛋白含有 </span> </p> </li> <li> <div> <b>5. </b><a class="enjiyixqcontent" href="/patent-detail/06130435340406.html">methods for screening for a therapeutic agent, for suppressing the polynucleotide sequence, for treating a non-steroidal cancer, for screening for binding of an agent specifically to a polynucleotide, and for determining whether a patient is at risk for developing or having a non-steroidal cancer, pharmaceutical composition, uses of a therapeutic agent, an antisense molecule or a cell that expresses and / or contains the antisense molecule, at least one of the immunogenic membrane proteins, fragments, derivatives or homologues thereof or from a cell containing and / or expressing at least one of the immunogenic membrane proteins or fragments, derivatives or homologues thereof and an agent or antibody, agent, and kit for identifying a patient at risk of developing or have non-steroidal cancer</a> <b>[P]</b> . <span> 外国专利： <!-- --> BRPI0414446A </span> <span> . 2006-11-14</span> </div> <p class="zwjiyix translation" style="max-width: initial;height: auto;word-break: break-all;white-space: initial;text-overflow: initial;overflow: initial;"> <span>机译：筛选治疗剂，抑制多核苷酸序列，治疗非甾体类癌症，筛选试剂特异性结合多核苷酸的方法以及确定患者是否处于发展或患有非糖尿病风险的方法类固醇癌，药物组合物，表达和/或含有反义分子的治疗剂，反义分子或细胞，免疫原性膜蛋白，其片段，衍生物或同源物中至少一种或来自含有和/或含有细胞的细胞的用途或表达至少一种免疫原性膜蛋白或其片段，衍生物或同系物以及一种试剂或抗体，试剂和试剂盒，用于鉴定有发生或患有非甾体癌风险的患者 </span> </p> </li> </ul> </div> </div> </div> <div class="theme cardcommon" style="overflow: auto;display:none"> <h3 class="all_title" id="enpatent55">相关主题</h3> <ul id="subject"> </ul> </div> </div> </div> </div> <div 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