A bicistronic vector with destabilized mRNA secondary structure yields scalable higher titer expression of human neurturin in        <i xmlns='http://www.wiley.com/namespaces/wiley'>E. coliE. coli

Roy Varnika; Roth Robert; Berge Mark; Chitta Rajesh; Vajrala Sucheta; Kuntumalla Srilatha; E. Schmelzer Albert; Schoner Ron

摘要

ABSTRACT <section xml:id="bit26299-sec-0001"> > Human neurturin (NTN) is a cystine knot growth factor with potential therapeutic use in diseases such as Parkinson's and diabetes. Scalable high titer production of native NTN is particularly challenging because of the cystine knot structure which consists of an embedded ring comprised of at least three disulfide bonds. We sought to pursue enhanced scalable production of NTN in Escherichia coli . Our initial efforts focused on codon optimization of the first two codons following AUG, but these studies resulted in only a marginal increase in NTN expression. Therefore, we pursued an alternative strategy of using a bicistronic vector for NTN expression designed to reduce mRNA secondary structure to achieve increased ribosome binding and re‐initiation. The first cistron was designed to prevent sequestration of the translation initiation region in a secondary conformation. The second cistron, which contained the NTN coding sequence itself, was engineered to disrupt double bonded base pairs and destabilize the secondary structure for ribosome re‐initiation. The ensemble approach of reducing NTN's mRNA secondary structure and using the bicistronic vector had an additive effect resulting in significantly increased NTN expression. Our strain selection studies were conducted in a miniaturized bioreactor. An optimized strain was selected and scaled up to a 100?L fermentor, which yielded an inclusion body titer of 2?g/L. The inclusion bodies were refolded to yield active NTN. We believe that our strategy is applicable to other candidate proteins that are difficult‐to‐express due to stable mRNA secondary structures. Biotechnol. Bioeng. 2017;114: 1753–1761. ? 2017 Wiley Periodicals, Inc. </section> </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 XMLNS =“http://www.wiley.com/namespaces/wiley”type =“main”xml：lang =“en”> <title type =“main”>抽象</ title> <section XML：ID =“Bit26299-SEC-0001”> <P>人神经蛋白（NTN）是一种胱氨酸结生长因子，具有潜在治疗用途，如帕金森和糖尿病。由于胱氨酸结结构，该天然NTN的可伸缩高滴度产生特别具有挑战性，该胱氨酸结结构包括由至少三种二硫键组成的嵌入环组成。我们试图在大肠杆菌中追求增强的NTN可扩展生产。我们的初步努力集中于八八次持续前两条密码子的密码子优化，但这些研究导致NTN表达的边际增加。因此，我们追求使用双顺调载体的替代策略对旨在降低mRNA二级结构以实现增加的核糖体结合和重新启动。设计第一Cistron以防止在二次构象中依赖翻译起始区域的封存。含有NTN编码序列本身的第二凯斯通设计成破坏双键基对对并使核糖体重新启动的二次结构变得破坏。减少NTN的mRNA二级结构和使用双顺调载体的集合方法具有显着增加的NTN表达产生的添加效果。我们的应变选择研究在小型化生物反应器中进行。选择优化的菌株并缩放到100·L发酵罐，其产生2μl掺杂剂的夹层。将包涵体重新折叠以产生活性NTN。我们认为，我们的策略适用于由于稳定的mRNA二级结构而难以明确的候选蛋白质。 Biotechnol。生物。 2017; 114：1753-1761。还2017 Wiley期刊，Inc。</ p> </ section> </ abstract> </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-15017/'>《Biotechnology and Bioengineering》</a> <b style="margin: 0 2px;">|</b><span>2017年第8期</span><b style="margin: 0 2px;">|</b><span>共9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=Roy Varnika&option=202" target="_blank" rel="nofollow">Roy Varnika;</a> <a href="/search.html?doctypes=4_5_6_1-0_4-0_1_2_3_7_9&sertext=Roth Robert&option=202" target="_blank" rel="nofollow">Roth Robert;</a> <a href="/search.html?doctypes=4_5_6_1-0_4-0_1_2_3_7_9&sertext=Berge Mark&option=202" target="_blank" rel="nofollow">Berge Mark;</a> <a href="/search.html?doctypes=4_5_6_1-0_4-0_1_2_3_7_9&sertext=Chitta Rajesh&option=202" target="_blank" rel="nofollow">Chitta Rajesh;</a> <a href="/search.html?doctypes=4_5_6_1-0_4-0_1_2_3_7_9&sertext=Vajrala Sucheta&option=202" target="_blank" rel="nofollow">Vajrala Sucheta;</a> <a href="/search.html?doctypes=4_5_6_1-0_4-0_1_2_3_7_9&sertext=Kuntumalla Srilatha&option=202" target="_blank" rel="nofollow">Kuntumalla Srilatha;</a> <a href="/search.html?doctypes=4_5_6_1-0_4-0_1_2_3_7_9&sertext=E. Schmelzer Albert&option=202" target="_blank" rel="nofollow">E. Schmelzer Albert;</a> <a href="/search.html?doctypes=4_5_6_1-0_4-0_1_2_3_7_9&sertext=Schoner Ron&option=202" target="_blank" rel="nofollow">Schoner Ron;</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>Cell Culture and Fermentation SciencesMedImmune LLC 1 MedImmune Way Gaithersburg Maryland 20878;</p> <p>Innovative MedicinesAstraZeneca R&</p> <p>DM?lndal Sweden;</p> <p>Cell Culture and Fermentation SciencesMedImmune LLC 1 MedImmune Way Gaithersburg Maryland 20878;</p> <p>Cell Culture and Fermentation SciencesMedImmune LLC 1 MedImmune Way Gaithersburg Maryland 20878;</p> <p>Cell Culture and Fermentation SciencesMedImmune LLC 1 MedImmune Way Gaithersburg Maryland 20878;</p> <p>Analytical BiotechnologyMedImmune LLCGaithersburg Maryland;</p> <p>Cell Culture and Fermentation SciencesMedImmune LLC 1 MedImmune Way Gaithersburg Maryland 20878;</p> <p>Cell Culture and Fermentation SciencesMedImmune LLC 1 MedImmune Way Gaithersburg Maryland 20878;</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/180.html" title="生物工程学（生物技术）">生物工程学（生物技术）;</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=neurturin&option=203" rel="nofollow">neurturin;</a> <a style="color: #3E7FEB;" href="/search.html?doctypes=4_5_6_1-0_4-0_1_2_3_7_9&sertext=cystine knot&option=203" rel="nofollow">cystine knot;</a> <a style="color: #3E7FEB;" href="/search.html?doctypes=4_5_6_1-0_4-0_1_2_3_7_9&sertext=mRNA secondary structure&option=203" rel="nofollow">mRNA secondary structure;</a> <a style="color: #3E7FEB;" href="/search.html?doctypes=4_5_6_1-0_4-0_1_2_3_7_9&sertext=destabilization&option=203" rel="nofollow">destabilization;</a> <a style="color: #3E7FEB;" href="/search.html?doctypes=4_5_6_1-0_4-0_1_2_3_7_9&sertext=bicistronic vector scalable production&option=203" rel="nofollow">bicistronic vector scalable production;</a> <a style="color: #3E7FEB;" href="/search.html?doctypes=4_5_6_1-0_4-0_1_2_3_7_9&sertext=higher titer&option=203" rel="nofollow">higher titer;</a> <a style="color: #3E7FEB;" href="/search.html?doctypes=4_5_6_1-0_4-0_1_2_3_7_9&sertext=E. coli&option=203" rel="nofollow">E. coli;</a> <a style="color: #3E7FEB;" href="/search.html?doctypes=4_5_6_1-0_4-0_1_2_3_7_9&sertext=Micro‐24&option=203" rel="nofollow">Micro‐24;</a> </p> <div class="translation"> 机译：神经蛋白;胱氨酸结;mRNA二级结构;稳定化;双顺反应载体可伸缩生产;较高的滴度;e。大肠杆菌;Micro-24; 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Polymer Physics .</a> <span>2017</span><span>,第21期</span> </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：纳米纤细polysulfone / tio <sub xmlns =“http://www.wiley.com/namespaces/wiley”> 2 </ sub> 2纳米复合材料：表面属性及其与<i xmlns =“的关系http：//www.wiley .com /命名空间/ wiley“> e。大肠杆菌</ i> e。 Coli粘附</span> </p> </li> <li> <div> <b>6. </b><a class="enjiyixqcontent" href="/journal-foreign-detail/0704025422909.html">Effects of metabolic engineering on downstream processing operational cost and energy consumption: the case of <fc xmlns="http://www.wiley.com/namespaces/wiley"> <fi>Escherichia coli</fi> </fc> <fi xmlns="http://www.wiley.com/namespaces/wiley">Escherichia coli</fi>Escherichia coli <fi xmlns="http://www.wiley.com/namespaces/wiley">'s</fi>'s glycerol conversion to succinic acid</a> <b>[J]</b> . <span> <a href="/search.html?doctypes=4_5_6_1-0_4-0_1_2_3_7_9&sertext=Tafur Rangel Albert Enrique&option=202" target="_blank" rel="nofollow" class="tuijian_auth tuijian_authcolor">Tafur Rangel Albert Enrique,</a> <a href="/search.html?doctypes=4_5_6_1-0_4-0_1_2_3_7_9&sertext=Camelo Valera Laura Carolina&option=202" target="_blank" rel="nofollow" class="tuijian_auth tuijian_authcolor">Camelo Valera Laura Carolina,</a> <a href="/search.html?doctypes=4_5_6_1-0_4-0_1_2_3_7_9&sertext=Gómez Ramírez Jorge Mario&option=202" target="_blank" rel="nofollow" class="tuijian_auth tuijian_authcolor">Gómez Ramírez Jorge Mario,</a> <a href="/journal-foreign-33908/" target="_blank" rel="nofollow" class="tuijian_authcolor">Journal of Chemical Technology & Biotechnology .</a> <span>2018</span><span>,第7期</span> </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：代谢工程对下游加工运营成本和能源消耗的影响：<FC XMLNS =“http://www.wiley.com/namespaces/wiley”> <fi> escherichia coli </ fi> </ fc> < fi xmlns =“http://www.wiley.com/namespace/wiley”> escherichia coli </ fi> escherichia coli <fi xmlns =“http://www.wiley.com/namespaces/wiley”>'s < / fi>对琥珀酸的甘油转化为琥珀酸</span> </p> </li> <li> <div> <b>7. </b><a class="enjiyixqcontent" href="/academic-conference-foreign_microbial-engineering_thesis/020512312996.html">LEARNING FROM THE MAMMALIAN EXPRESSION SYSTEM TO DEVELOP A HIGH TITER HALF-ANTIBODY PROCESS IN E. COLI</a> <b>[C]</b> . <span> <a href="/search.html?doctypes=4_5_6_1-0_4-0_1_2_3_7_9&sertext=Emily Dong&option=202" target="_blank" rel="nofollow" class="tuijian_auth tuijian_authcolor">Emily Dong </a> <a href="/conference-foreign-46662/" target="_blank" rel="nofollow" class="tuijian_authcolor">Microbial engineering .</a> <span>2018</span> </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：从哺乳动物表达系统中学习发展大肠杆菌中的高滴度半抗原过程</span> </p> </li> <li> <div> <b>8. </b><a class="enjiyixqcontent" href="/academic-journal-foreign-pmc_detail_thesis/040001848383.html">The influence of mRNA primary and secondary structure on human IFN-gamma gene expression in E. coli.</a> <b>[O] </b> . <span> <a href="/search.html?doctypes=4_5_6_1-0_4-0_1_2_3_7_9&sertext=L H Tessier&option=202" target="_blank" rel="nofollow" class="tuijian_auth tuijian_authcolor">L H Tessier,</a> <a href="/search.html?doctypes=4_5_6_1-0_4-0_1_2_3_7_9&sertext=P Sondermeyer&option=202" target="_blank" rel="nofollow" class="tuijian_auth tuijian_authcolor">P Sondermeyer,</a> <a href="/search.html?doctypes=4_5_6_1-0_4-0_1_2_3_7_9&sertext=T Faure&option=202" target="_blank" rel="nofollow" class="tuijian_auth tuijian_authcolor">T Faure,</a> <span>1984</span> </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：mRNA一级和二级结构对大肠杆菌中人IFN-γ基因表达的影响。</span> </p> </li> <li> <div> <b>9. </b><a class="enjiyixqcontent" href="/open-access_resources_thesis/01000127735192.html">Figure 8: Codon optimization and mRNA structure of CoV-RMEN gene for expression in E. coli.</a> <b>[O] </b> . <span> <a href="/search.html?doctypes=4_5_6_1-0_4-0_1_2_3_7_9&sertext=&option=202" target="_blank" rel="nofollow" class="tuijian_auth tuijian_authcolor"> </a> <span>-1</span> </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：图8：CoV-RMen基因的Codon优化和MRNA结构在大肠杆菌中表达的表达。</span> </p> </li> <li> <div> <b>10. </b><a class="enjiyixqcontent" href="/ntis-science-report_ad_thesis/020711187449.html">Expression and Refolding of Functional Human Butyrylcholinesterase from E. Coli</a> <b>[R] </b> . <span> <a href="/search.html?doctypes=4_5_6_1-0_4-0_1_2_3_7_9&sertext=Masson, P.&option=202" target="_blank" rel="nofollow" class="tuijian_auth tuijian_authcolor">Masson, P.,</a> <a href="/search.html?doctypes=4_5_6_1-0_4-0_1_2_3_7_9&sertext=Adkins, S.&option=202" target="_blank" rel="nofollow" class="tuijian_auth tuijian_authcolor">Adkins, S.,</a> <a href="/search.html?doctypes=4_5_6_1-0_4-0_1_2_3_7_9&sertext=Pham-Trong, P.&option=202" target="_blank" rel="nofollow" class="tuijian_auth tuijian_authcolor">Pham-Trong, P.,</a> <span>1992</span> </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：大肠杆菌功能性人丁酰胆碱酯酶的表达和重折叠</span> </p> </li> </ul> <ul style="display: none;"> <li> <div> <b>1. </b><a class="enjiyixqcontent" href="/academic-journal-cn_chinese-science_thesis/0201227492868.html">Expression of human transforming growth factor β1 in E. coli (I) --Direct expression in cytoplasm</a> <b>[J]</b> <span> <a href="/search.html?doctypes=4_5_6_1-0_4-0_1_2_3_7_9&sertext=龙建银&option=202" target="_blank" rel="nofollow" class="tuijian_auth tuijian_authcolor"> . 龙建银</a> <a href="/search.html?doctypes=4_5_6_1-0_4-0_1_2_3_7_9&sertext=王会信&option=202" target="_blank" rel="nofollow" class="tuijian_auth tuijian_authcolor">,王会信</a> <a href="/search.html?doctypes=4_5_6_1-0_4-0_1_2_3_7_9&sertext=刘莉&option=202" target="_blank" rel="nofollow" class="tuijian_auth tuijian_authcolor">,刘莉</a> <span> <a href="/journal-cn-10213/" target="_blank" rel="nofollow" class="tuijian_authcolor"> . 中国科学 </a> </span> <span> . 1998</span><span>,第005期</span> </span> </div> </li> <li> <div> <b>2. </b><a class="enjiyixqcontent" href="/academic-journal-cn_chinese-science_thesis/0201227512251.html">MOLECULAR CLONING OF A NEW VARIANT OF HUMAN PAPILLOMAVIRUS TYPE 6 ISOLATED FROM A CHINESE WOMAN AND EXPRESSION OF ITS L1 GENE IN E. coli——MOLECULAR CLONING EXPRESSION OF HPV6 GENE</a> <b>[J]</b> <span> <a href="/search.html?doctypes=4_5_6_1-0_4-0_1_2_3_7_9&sertext=舒琍琍&option=202" target="_blank" rel="nofollow" class="tuijian_auth tuijian_authcolor"> . 舒琍琍</a> <a href="/search.html?doctypes=4_5_6_1-0_4-0_1_2_3_7_9&sertext=冯慧敏&option=202" target="_blank" rel="nofollow" class="tuijian_auth tuijian_authcolor">,冯慧敏</a> <a href="/search.html?doctypes=4_5_6_1-0_4-0_1_2_3_7_9&sertext=金奇&option=202" target="_blank" rel="nofollow" class="tuijian_auth tuijian_authcolor">,金奇</a> <span> <a href="/journal-cn-10215/" target="_blank" rel="nofollow" class="tuijian_authcolor"> . 中国科学 </a> </span> <span> . 1992</span><span>,第005期</span> </span> </div> </li> </ul> <ul style="display: none;"> <li> <div> <b>1. </b><a class="enjiyixqcontent" href="/patent-detail/06130446111865.html">-16 -16 FUSION PROTEIN OF TAG PROTEIN AND HUMAN INTERLEUKIN-16 GENE ENCODING SAID FUSION PROTEIN EXPRESSION VECTOR CONTAINING SAID GENE E. COLI TRANSFORMED WITH SAID VECTOR AND PROCESS FOR PRODUCING HUMAN INTERLEUKIN-16 USING SAID E. COLI</a> <b>[P]</b> . <span> 外国专利：  KR20020003917A </span> <span> . 2002-01-16</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：-16 -16包含标签蛋白和人类白细胞介素16基因的融合蛋白，其中包含含有所说基因E. SAIL的人工融合蛋白表达载体，并利用所述大肠杆菌转化了表达人类白介素16的过程。 </span> </p> </li> <li> <div> <b>2. </b><a class="enjiyixqcontent" href="/patent-detail/06130458955184.html">Microbial hybrid promoter / operator, and the expression vector containing the same, and E. coli strain transformed with the expression vector, culture of E. coli strain and process for the microbial production of heterologous polypeptides using the culture</a> <b>[P]</b> . <span> 外国专利：  DK170477B1 </span> <span> . 1995-09-11</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：微生物杂种启动子/操纵子，以及包含该杂种启动子/操纵子的表达载体，以及用该表达载体转化的大肠杆菌菌株，大肠杆菌菌株的培养以及使用该培养基进行微生物生产异源多肽的方法 </span> </p> </li> <li> <div> <b>3. </b><a class="enjiyixqcontent" href="/patent-detail/06130403036017.html">GENETIC CONSTRUCT BASED ON TWO INDUCIBLE EXPRESSION VECTORS FOR EXPRESSION OF THIAZOLE/OXAZOLE MODIFIED PEPTIDE IN CELLS OF BACTERIA E. COLI; METHOD FOR PRODUCING RECOMBINANT STRAIN OF BACTERIA E. COLI AND METHOD FOR PRODUCING MODIFIED PEPTIDE BASED THEREOF</a> <b>[P]</b> . <span> 外国专利：  RU2694044C2 </span> <span> . 2019-07-08</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：基于两个诱导表达载体的基因构建用于在大肠杆菌中表达噻唑/恶唑修饰的肽;产大肠杆菌重组菌株的方法及基于其的修饰肽的制备方法 </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 class="right rightcon"> <div class="details_img cardcommon clearfix" style="margin-bottom: 10px;display:none;" > </div> </div> </div> <div id="thesis_get_original1" class="downloadBth" style="bottom: 19px;z-index: 999;" onclick="ywcd('0704028288254','4',7,2,1,'',this,24)" class="delivery" prompt="010401" title="通过人工服务将文献原文发送至邮箱" >获取原文</div> <div class="journalsub-pop-up" style="display: none"> <div 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A bicistronic vector with destabilized mRNA secondary structure yields scalable higher titer expression of human neurturin in E. coliE. coli

摘要