Heterologous expression, purification, and functional characterization of recombinant ovine angiotensinogen in the methylotrophic yeast Pichia pastoris

Palanikumar Indumathi; Katla Srikanth; Tahara Nariyasu; Yui Midori; Zhang Rui; Ebihara Akio; Sivaprakasam Senthilkumar

摘要

Abstract > Angiotensinogen (AGT), a glycosylated plasma noninhibitory serpin, serves as a precursor for angiotensin peptides which regulate blood pressure and electrolyte balance. AGT is specifically cleaved by renin to produce angiotensin‐I, the first product of the angiotensin‐processing cascade. Ovine angiotensinogen (oAGT) is considered an effective substrate for human renin and consequently finds application in clinical renin assays. In this study, oAGT was cloned into the genome of Pichia pastoris and expressed under the control of alcohol oxidase (AOX1) promoter for high‐level production. Compared to the shake flask study, the high cell density cultivation in bioreactor resulted in multifold increase in oAGT titer (420?±?9.26?mg/L), which is its highest reported titer to date. We purified recombinant oAGT to homogeneity using two chromatography steps. The characterization studies revealed oAGT underwent a two‐state transition during thermal denaturation process as assessed by differential scanning fluorimetry, and the melting temperature ( T <sub>m</sub> ) of the purified oAGT from P. pastoris was 48.3°C. Renin reactivity with recombinant oAGT from P. pastoris (0.51?nM angiotensin‐I/min) was slightly lower than the renin reactivity for recombinant oAGT from <fi>Escherichia coli</fi> (0.67?nM angiotensin‐I/min), possibly because of its mannosylated N‐glycan content. Enhanced production of functionally active recombinant oAGT using P. pastoris expression system reported in this study envisage the effective utilization of oAGT in clinical studies related to renin in near future. </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> > 血管紧张素素（AGT）是一种糖基化的血浆非抑制蛇素，用作调节血压和电解质平衡的血管紧张素肽的前体。肾素特异性地切割，以产生血管紧张素-I，该血管紧张素加工级联的第一产物。绵羊血管紧张素（OAGT）被认为是人类肾素的有效底物，因此在临床肾素测定中发现应用。在这项研究中，OAGT被克隆到基因组中 Pichia Pastoris </ i> 并在醇氧化酶（AXOX1）启动子的控制下表达，用于高级别生产。与摇瓶研究相比，生物反应器中的高细胞密度培养导致OAGT滴度（420？±9.26×9.26×mg / L）中的多变升高，这是其最高报告的迄今为止。我们使用两种色谱步骤纯化重组OAG到均匀性。表征研究显示，由于差示扫描荧光测定法和熔化温度（ t </ i> <sub> m </ sub> ）来自纯化的OAGT p。 Pastoris </ i> 是48.3°C。与重组OAGT的肾素反应性 p。 Pastoris </ i> （0.51？NM血管紧张素-I / min）略低于RENON反应性的重组卵石 <fi>大肠杆菌</ fi> （0.67？NM血管紧张素-I / min），可能是因为其甘露糖基化的N-聚糖含量。增强了功能性主动重组织布的生产使用 p。 Pastoris </ i> 本研究报告的表达系统设想了OAGT在不久的将来与肾素相关的临床研究中的有效利用。 </ 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5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=Palanikumar Indumathi&option=202" target="_blank" rel="nofollow">Palanikumar Indumathi;</a> <a href="/search.html?doctypes=4_5_6_1-0_4-0_1_2_3_7_9&sertext=Katla Srikanth&option=202" target="_blank" rel="nofollow">Katla Srikanth;</a> <a href="/search.html?doctypes=4_5_6_1-0_4-0_1_2_3_7_9&sertext=Tahara Nariyasu&option=202" target="_blank" rel="nofollow">Tahara Nariyasu;</a> <a href="/search.html?doctypes=4_5_6_1-0_4-0_1_2_3_7_9&sertext=Yui Midori&option=202" target="_blank" rel="nofollow">Yui Midori;</a> <a href="/search.html?doctypes=4_5_6_1-0_4-0_1_2_3_7_9&sertext=Zhang Rui&option=202" target="_blank" rel="nofollow">Zhang Rui;</a> <a href="/search.html?doctypes=4_5_6_1-0_4-0_1_2_3_7_9&sertext=Ebihara Akio&option=202" target="_blank" rel="nofollow">Ebihara Akio;</a> <a href="/search.html?doctypes=4_5_6_1-0_4-0_1_2_3_7_9&sertext=Sivaprakasam Senthilkumar&option=202" target="_blank" rel="nofollow">Sivaprakasam Senthilkumar;</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>BioPAT LaboratoryIndian Institute of Technology GuwahatiGuwahati Assam India;</p> <p>BioPAT LaboratoryIndian Institute of Technology GuwahatiGuwahati Assam India;</p> <p>Graduate School of Applied Biological SciencesGifu UniversityGifu Japan;</p> <p>Graduate School of Applied Biological SciencesGifu UniversityGifu Japan;</p> <p>Faculty of Applied Biological SciencesGifu UniversityGifu Japan;</p> <p>Faculty of Applied Biological SciencesGifu UniversityGifu Japan;</p> <p>BioPAT LaboratoryIndian Institute of Technology GuwahatiGuwahati Assam India;</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=alcohol oxidase&option=203" rel="nofollow">alcohol oxidase;</a> <a style="color: #3E7FEB;" href="/search.html?doctypes=4_5_6_1-0_4-0_1_2_3_7_9&sertext=high cell density cultivation&option=203" rel="nofollow">high cell density cultivation;</a> <a style="color: #3E7FEB;" href="/search.html?doctypes=4_5_6_1-0_4-0_1_2_3_7_9&sertext=ovine angiotensinogen&option=203" rel="nofollow">ovine angiotensinogen;</a> <a style="color: #3E7FEB;" href="/search.html?doctypes=4_5_6_1-0_4-0_1_2_3_7_9&sertext=Pichia pastoris&option=203" rel="nofollow">Pichia pastoris;</a> </p> <div class="translation"> 机译：醇氧化酶;高细胞密度栽培;绵羊血管紧张素;Pichia Pastoris; </div> </li> </ul> </div> </div> <div class="literature cardcommon"> <div class="similarity "> 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Tatsuro Shibui</a> <a href="/search.html?doctypes=4_5_6_1-0_4-0_1_2_3_7_9&sertext=Keisuke Bando&option=202" target="_blank" rel="nofollow" class="tuijian_auth tuijian_authcolor">,Keisuke Bando</a> <a href="/search.html?doctypes=4_5_6_1-0_4-0_1_2_3_7_9&sertext=Satoru Misawa&option=202" target="_blank" rel="nofollow" class="tuijian_auth tuijian_authcolor">,Satoru Misawa</a> <span> <a href="/journal-cn-56588/" target="_blank" rel="nofollow" class="tuijian_authcolor"> . 生命科学与技术进展（英文） </a> </span> <span> . 2013</span><span>,第5期</span> </span> </div> </li> <li> <div> <b>4. </b><a class="enjiyixqcontent" href="/academic-journal-cn_chinese-biology-abstracts_thesis/0201255429487.html">Cloning and expression of Thermobifida xylanase gene in the methylotrophic yeast Pichia pastoris</a> <b>[J]</b> <span> <a href="/search.html?doctypes=4_5_6_1-0_4-0_1_2_3_7_9&sertext=Cheng&option=202" target="_blank" rel="nofollow" class="tuijian_auth tuijian_authcolor"> . 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GONG Xing-guo(龚兴国)</a> <a href="/search.html?doctypes=4_5_6_1-0_4-0_1_2_3_7_9&sertext=ZHONG Wen-tao(钟文涛)&option=202" target="_blank" rel="nofollow" class="tuijian_auth tuijian_authcolor">,ZHONG Wen-tao(钟文涛)</a> <a href="/search.html?doctypes=4_5_6_1-0_4-0_1_2_3_7_9&sertext=Xie Jie(谢捷)&option=202" target="_blank" rel="nofollow" class="tuijian_auth tuijian_authcolor">,Xie Jie(谢捷)</a> <span> <a href="/conference-cn-28435/" target="_blank" rel="nofollow" class="tuijian_authcolor"> . 首届长三角科技论坛：长三角生物医药发展论坛 </a> <span> <span> . 2004</span> </span> </div> </li> <li> <div> <b>6. </b><a class="enjiyixqcontent" href="/academic-degree-domestic_mphd_thesis/020311530984.html">Biosynthesis of Silver Nanoparticles by sundried Aleurites moluccana leaf and yeast pichia pastoris</a> <b>[A] </b> <span> <a href="/search.html?doctypes=4_5_6_1-0_4-0_1_2_3_7_9&sertext=Egesa Dan&option=202" target="_blank" rel="nofollow" class="tuijian_auth tuijian_authcolor"> . Egesa Dan</a> <span> . 2009</span> </span> </div> </li> </ul> <ul style="display: none;"> <li> <div> <b>1. </b><a class="enjiyixqcontent" href="/patent-detail/06130405777754.html">RECOMBINANT PLASMID FOR EXPRESSION IN PICHIA PASTORIS YEAST GENE OF CHIMERIC PROTEIN OF HUMAN ANGIOGENIN AND STRAIN OF PICHIA PASTORIS YEAST - PRODUCER OF RECOMBINANT CHIMERIC PROTEIN OF HUMAN ANGIOGENIN</a> <b>[P]</b> . <span> 外国专利：  RU2658758C1 </span> <span> . 2018-06-22</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：重组质粒在人血管生成素嵌合蛋白的Pichia Pastoris酵母基因中的表达及Pichia Pastoris酵母的菌株-重组人血管生成素的嵌合蛋白。 </span> </p> </li> <li> <div> <b>2. </b><a class="enjiyixqcontent" href="/patent-detail/06130400516121.html">RECOMBINANT PLASMID DNA pPicZαA/BChE-14 CONTAINING HUMAN MODIFIED BUTYRYLCHOLINESTERASE GENE FOR EXPRESSION OF BUTYRYLCHOLINESTERASE GENE IN METHYLOTROPHIC YEAST PICHIA PASTORIS FOR THERAPY OF ORGANOPHOSPHOROUS TOXINS POISONING</a> <b>[P]</b> . <span> 外国专利：  RU2711939C2 </span> <span> . 2020-01-23</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：重组人质粒修饰的酪氨酰胆碱酯酶基因的质粒质粒pPicZαA/ BChE-14在甲基营养型酵母菌毕赤酵母中表达酪氨酰胆碱酯酶基因，用于治疗有机磷毒素中毒 </span> </p> </li> <li> <div> <b>3. </b><a class="enjiyixqcontent" href="/patent-detail/06130405819194.html">RECOMBINANT PLASMID DNA pPicZαA/BChE-14 CONTAINING HUMAN MODIFIED BUTYRYLCHOLINESTERASE GENE FOR EXPRESSION OF BUTYRYLCHOLINESTERASE GENE IN METHYLOTROPHIC YEAST PICHIA PASTORIS FOR THERAPY OF ORGANOPHOSPHOROUS TOXINS POISONING</a> <b>[P]</b> . <span> 外国专利：  RU2016152261A3 </span> <span> . 2018-07-02</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：重组修饰质粒pPicZαA/ 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