המחברים מודים לד&quot;ר תומס Rademacher למתן וקטור ppam צמח הביטוי ואבראהים אל עמדי לטיפוח צמחי טבק המשמשים במחקר זה. ברצוננו להודות לד&quot;ר ריצ&#39;רד מ Twyman לסיועו בעריכת כתב היד. עבודה זו בחלקו מומנה על ידי המועצה האירופית למחקר מתקדם גרנט &quot;העתיד-פארמה&quot;, מספר הצעת 269,110 וFraunhofer Zukunftsstiftung (Fraunhofer עתיד הקרן).

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אגרת הפרסום מומנה באופן חלקי על ידי חברות Statease, Inc (ארה&quot;ב) וStatcon (גרמניה), שלא היו מעורבות במעורב בהכנת כתב היד או אחראית לכל התוכן שלו.

כל ניסוי דורש תכנון זהיר, כי משאבים הם לרוב נדירים ויקרים. זה נכון במיוחד לאסטרטגיות מחלקת אנרגיה בגלל שגיאות במהלך שלב תכנון (לדוגמא: בחירת דגם בסיס שאינו מכסה את כל אינטראקציות הגורם משמעותיות) באופן משמעותי יכולות לצמצם את כוח הניבוי של מודלים וכתוצאה מכך ובכ?...

הייצור של חלבונים ביולוגיות בצמחים יש יתרון משום שצמחים הם זולים לגדול, הפלטפורמה וניתן לשנותם רק על ידי גידול צמחים יותר, ופתוגנים אנושיים אינם מסוגלים לשכפל 1,2. אסטרטגיות ביטוי חולפות מבוססות למשל על החדירה של עלים עם Agrobacterium tumefaciens מספקת יתרונות נוספים בגלל הזמן בין נקודת מסירת DNA והמשלוח של מוצר מטוהר מצטמצם משנה לפחות מ 2 חודשים 3. ביטוי חולף משמש גם לניתוח פונקציונלי, למשל כדי לבדוק גנים ביכולתם להשלים את מוטציות אובדן של פונקציה או לחקור אינטראקציות חלבון 4-6. עם זאת, רמות ביטוי חולפות נוטות להראות גדולה יותר וריאציה אצווה כדי אצווה מ רמות ביטוי בצמחים מהונדסים 7-9. הדבר זה מפחית את הסבירות לכך שתהליכי ייצור ביולוגיות המבוססים על wi ביטוי החולףתהיה מאושר בהקשר של תרגול טוב ייצור (GMP), כי שחזור הוא תכונת איכות קריטית והוא כפוף להערכת סיכונים 10. וריאציה כזו יכולה גם להסוות את כל אינטראקציות שחוקרים מתכוונים לחקור. לכן, יצאנו לזהות את הגורמים העיקריים המשפיעים על רמות ביטוי חולפות בצמחים ולבנות מודל חיזוי כמותית באיכות גבוהה. הגישה אחת גורם ב-פעמי (OFAT) משמשת לעתים קרובות כדי לאפיין את ההשפעה (אפקט) של פרמטרים מסוימים (גורמים) על התוצאה (בתגובה) של ניסוי 11. אבל זה לא טובים, כי הבדיקות בודדות (פועל) במהלך חקירה (ניסוי) תהיה מיושרת כמו פנינים במחרוזת דרך אזור הפוטנציאל מורחב על ידי הגורמים שנבדקו (מרחב תכנון). הכיסוי של עיצוב החלל, ולכן המידה של מידע ההופק מהניסוי הואדל, כפי שמוצג באיור 1 א 12. יתר על כן, תלות הדדית בין גורמים שונים (אינטראקציות גורם) יכולה להישאר נסתרת וכתוצאה מכך מודלים עניים ו / או החיזוי של אופטימה הכוזבת, כפי שמוצג באיור 1 ב 13. ניתן להימנע מהחסרונות שתוארו לעיל באמצעות עיצוב של ניסויי גישה (DOE), שבריצות של ניסוי מפוזרות בצורה שווה יותר לאורך כל מרחב תכנון, כלומר יותר מגורם אחד הוא נע בין שתי ריצות 14. ישנם עיצובים מיוחדים לתערובות, הקרנת גורמים (עיצובי עצרת) ולכמת את השפעות גורם בתגובות (שיטות משטח תגובה, RSM ים) 15. יתר על כן, יכול להתממש RSMs עיצובים מרכזיים-מרוכבים אבל גם יכול להיות מושגת בצורה יעילה על ידי שימוש בתוכנות מיוחדות שניתן להחיל קריטריונים שונים לבחירה של ריצות. לדוגמא, מה שנקרא D-optimalitקריטריון y יבחר ריצות כך כדי למזער את השגיאה במקדמים של המודל וכתוצאה מכך, בעוד שקריטריון IV-האופטימלי בוחר ריצות כי להשיג את שונות התחזית הנמוכה ביותר בחלל העיצוב 15,16. RSM אנו מתארים כאן מאפשר לכמת המדויקים של ביטוי חלבון החולף בצמחים, אבל זה יכול בקלות להיות מועבר לכל מערכת מעורבת מספר (~ 5-8) גורמים מספריים (למשל טמפרטורה, זמן, ריכוז) וכמה (~ 2 - 4) גורמים משמעיים (למשל אמרגן, צבע) שבו תיאור מכניסטית אינו זמין או מורכב מדי לדגמן. גישת מחלקת האנרגיה שמקורה במדעי החקלאות, אך התפשטה לאזורים אחרים בגלל זה הוא להעברה לכל מצב שבו כדאי להקטין את מספר ריצות צורך לקבל נתונים אמינים וליצור מודלים תיאורים עבור תהליכים מורכבים. זה בתורו הוביל להכללתה של מחלקת אנרגיה ב&quot; ההדרכה לתעשייה, Q8 (R2) תרופות פיתוח &quot;, שפורסם על ידי הרמוני הבינלאומי של דרישות טכניות עבור רישום של תרופות לשימוש בבני אדם (ICH) 17. DOE הוא כיום בשימוש נרחב במחקר ותעשייה 18 מדעיים. עם זאת, יש להקפיד במהלך התכנון והביצוע של הניסוי, כי בחירת תואר פולינום לא תקין למודל מרובה ליניארי רגרסיה (דגם בסיס) יכולים להציג את צורך בריצות נוספות למודל את כל השפעות הגורם בצורה נכונה. יתר על כן, פגום או חסר נתונים ליצור מודלים ופגומים שגויים תחזיות, ואף עלול למנוע כל ניסיון בניית מודל כפי שמתואר בסעיפי הפרוטוקול ודיון 18. בקטע הפרוטוקול, יהיה להגדיר תחילה אנו את צעדי תכנון החשובים ביותר לניסוי מבוסס RSM ולאחר מכן להסביר את העיצוב מבוסס על האיילה v8.1 תוכנת DesignExpert. אבל עיצובים דומים יכול להיות בנוי עם includi תוכנה אחרng JMP, Modde, וSTATISTICA. הליכי הניסוי ואחריו הוראות לניתוח נתונים והערכה. <img alt="איור 1" fo:content-width="5in" fo:src="/files/ftp_upload/51216/51216fig1highres.jpg" src="/files/ftp_upload/51216/51216fig1.jpg" /> איור 1. השוואה של OFAT והאיילה.. וריאציה רציפה של גורם אחד בכל פעם (OFAT) בניסוי (עיגולים שחורים, אדום וכחול) משיגה כיסוי נמוך של מרחב תכנון (אזורים בקעו). לעומת זאת, הווריאציה של יותר מגורם אחד בכל פעם באמצעות העיצוב של ניסויי אסטרטגיה (DOE) (עיגולים ירוקים) משפרת את הכיסוי ובכך הדיוק של המודלים וכתוצאה מכך. ב &#39;. כיסוי מרחב תכנון המוטה אומר שניסויי OFAT (עיגולים שחורים) גם יכולים שלא לזהות את האזורים אופטימליים הפעלה (אדום) ולחזות פתרונות לא אופטימליים (עיגול שחור גדול), ואילו strategi מחלקת האנרגיהes (כוכבים שחורים) יש סיכוי גבוה יותר לזהות תנאים עדיפים (כוכב שחור גדול).

<table><tbody><tr><td>Design-Expert(R) 8</td><td>Stat-Ease, Inc.</td><td>n.a.</td><td>DoE software</td></tr><tr><td>Tryptone</td><td>Carl Roth GmbH</td><td>8952.2</td><td>Media component</td></tr><tr><td>Yeast extract</td><td>Carl Roth GmbH</td><td>2363.2</td><td>Media component</td></tr><tr><td>Sodium chloride</td><td>Carl Roth GmbH</td><td>P029.2</td><td>Media component</td></tr><tr><td>Ampicillin</td><td>Carl Roth GmbH</td><td>K029.2</td><td>Antibiotic</td></tr><tr><td>Agar-Agar</td><td>Carl Roth GmbH</td><td>5210.2</td><td>Media component</td></tr><tr><td>&lt;i&gt;Escherichia coli&lt;/i&gt; K12 DH5a</td><td>Life Technologies</td><td>18263-012</td><td>Microorganism</td></tr><tr><td>pPAM</td><td>GenBank</td><td>AY027531</td><td>Cloning/expression vector;&amp;nbsp;</td></tr><tr><td>NucleoSpin Plasmid&amp;nbsp;</td><td>MACHEREY-NAGEL GmbH</td><td>740588.250</td><td>Plasmid DNA isolation kit</td></tr><tr><td>NucleoSpin Gel and PCR Clean-up</td><td>MACHEREY-NAGEL GmbH</td><td>740609.250</td><td>Plasmid DNA purification kit</td></tr><tr><td>NanoDrop 2000</td><td>Thermo Scientific</td><td>n.a.</td><td>Spectrophotometer</td></tr><tr><td>NcoI</td><td>New England Biolabs Inc.</td><td>R3193L</td><td>Restrictionendonuclease</td></tr><tr><td>EcoRI</td><td>New England Biolabs Inc.</td><td>R3101L</td><td>Restrictionendonuclease</td></tr><tr><td>AscI</td><td>New England Biolabs Inc.</td><td>R0558L</td><td>Restrictionendonuclease</td></tr><tr><td>NEB 4</td><td>New England Biolabs Inc.</td><td>B7004S</td><td>Restrictionendonuclease buffer</td></tr><tr><td>TRIS</td><td>Carl Roth GmbH</td><td>4855.3</td><td>Media component</td></tr><tr><td>Disodium tetraborate</td><td>Carl Roth GmbH</td><td>4403.3</td><td>Media component</td></tr><tr><td>EDTA</td><td>Carl Roth GmbH</td><td>8040.2</td><td>Media component</td></tr><tr><td>Agarose</td><td>Carl Roth GmbH</td><td>6352.4</td><td>Media component</td></tr><tr><td>Bromophenol blue</td><td>Carl Roth GmbH</td><td>A512.1</td><td>Color indicator</td></tr><tr><td>Xylene cyanol</td><td>Carl Roth GmbH</td><td>A513.1</td><td>Color indicator</td></tr><tr><td>Glycerol</td><td>Carl Roth GmbH</td><td>7530.2</td><td>Media component</td></tr><tr><td>Mini-Sub Cell GT Cell</td><td>BioRad</td><td>170-4406</td><td>Gel electrophoresis chamber</td></tr><tr><td>&lt;i&gt;Agrobacterium tumefaciens&lt;/i&gt; strain GV3101:pMP90RK</td><td>DSMZ</td><td>12365</td><td>Microorganism</td></tr><tr><td>Electroporator 2510</td><td>Eppendorf</td><td>4307000.658</td><td>Electroporator</td></tr><tr><td>Beef extract</td><td>Carl Roth GmbH</td><td>X975.2</td><td>Media component</td></tr><tr><td>Peptone</td><td>Carl Roth GmbH</td><td>2365.2</td><td>Media component</td></tr><tr><td>Sucrose</td><td>Carl Roth GmbH</td><td>4621.2</td><td>Media component</td></tr><tr><td>Magnesium sulfate</td><td>Carl Roth GmbH</td><td>0261.3</td><td>Media component</td></tr><tr><td>Carbenicillin</td><td>Carl Roth GmbH</td><td>6344.2</td><td>Antibiotic</td></tr><tr><td>Kanamycin</td><td>Carl Roth GmbH</td><td>T832.3</td><td>Antibiotic</td></tr><tr><td>Rifampicin</td><td>Carl Roth GmbH</td><td>4163.2</td><td>Antibiotic</td></tr><tr><td>FWD primer</td><td>Eurofins MWG Operon</td><td>n.a.</td><td>CCT CAG GAA GAG CAA TAC</td></tr><tr><td>REV primer</td><td>Eurofins MWG Operon</td><td>n.a.</td><td>CCA AAG CGA GTA CAC AAC</td></tr><tr><td>2720 Thermal cycler</td><td>Applied Biosystems</td><td>4359659</td><td>Thermocycler</td></tr><tr><td>RNAfold webserver</td><td>University of Vienna</td><td>n.a.</td><td>Software</td></tr><tr><td>Ferty 2 Mega</td><td>Kammlott</td><td>5.220072</td><td>Fertilizer</td></tr><tr><td>Grodan Rockwool Cubes 10 x10 cm</td><td>Grodan</td><td>n.a.</td><td>Rockwool block</td></tr><tr><td>Greenhouse</td><td>n.a.</td><td>n.a.</td><td>For plant cultivation</td></tr><tr><td>Phytotron</td><td>Ilka Zell</td><td>n.a.</td><td>For plant cultivation</td></tr><tr><td>Omnifix-F Solo</td><td>B. Braun</td><td>6064204</td><td>Syringe</td></tr><tr><td>Murashige and Skoog salts</td><td>Duchefa</td><td>M 0222.0010</td><td>Media component</td></tr><tr><td>Glucose</td><td>Carl Roth GmbH</td><td>6780.2</td><td>Media component</td></tr><tr><td>Acetosyringone</td><td>Sigma-Aldrich</td><td>D134406-5G</td><td>Phytohormon analogon</td></tr><tr><td>&amp;nbsp;BioPhotometer plus</td><td>Eppendorf</td><td>6132 000.008</td><td>Photometer</td></tr><tr><td>Osram cool white 36 W</td><td>Osram</td><td>4930440</td><td>Light source</td></tr><tr><td>Disodium phosphate</td><td>Carl Roth GmbH</td><td>4984.3</td><td>Media component</td></tr><tr><td>Centrifuge 5415D</td><td>Eppendorf</td><td>5424 000.410</td><td>Centrifuge</td></tr><tr><td>Forma -86C ULT freezer</td><td>ThermoFisher</td><td>88400</td><td>Freezer</td></tr><tr><td>Synergy HT</td><td>BioTek</td><td>SIAFRT</td><td>Fluorescence plate reader</td></tr><tr><td>Biacore T200</td><td>GE Healthcare</td><td>n.a.</td><td>SPR device</td></tr><tr><td>Protein A</td><td>Life Technologies</td><td>10-1006</td><td>Antibody binding protein</td></tr><tr><td>HEPES</td><td>Carl Roth GmbH</td><td>9105.3</td><td>Media component</td></tr><tr><td>Tween-20</td><td>Carl Roth GmbH</td><td>9127.3</td><td>Media component</td></tr><tr><td>2G12 antibody</td><td>Polymun</td><td>AB002</td><td>Reference antibody</td></tr></tbody></table>

characterization of complex systems using the design of experiments approach: transient protein expression in tobacco as a case study

צמחים מספקים יתרונות רבים לייצור תרופות ביולוגיים כוללים עלויות נמוכות, יכולת הרחבה, ובטיחות. ביטוי חולף מציע יתרון נוסף של פיתוח קצר וזמני ייצור, אבל רמות הביטוי יכולות להשתנות באופן משמעותי בין קבוצות ובכך הולידו חששות רגולטורים בהקשר של ייצור נאות. אנחנו השתמשנו בעיצוב של גישת ניסויים (DOE), כדי לקבוע את ההשפעה של גורמים מרכזיים כגון אלמנטים רגולטוריים במבנה הביטוי, גידול צמחים ופרמטרי פיתוח, ותנאי הדגירה בהבעה, בשונות של ביטוי בין קבוצות. בדקנו צמחים המבטאים נוגדן מודל אנטי HIV חד שבטי (2G12) וחלבון פלואורסצנטי סמן (DsRed). אנו דנים את הרציונל לבחירת מאפיינים מסוימים של המודל ולזהות מגבלות הפוטנציאל שלה. הגישה הכללית בקלות ניתן להעביר לבעיות אחרות, כי את עקרונות המודלמחדש החלים רחב: בחירת פרמטר המבוסס על ידע, הפחתת מורכבות על ידי פיצול הבעיה הראשונית לתוך מודולים קטנים יותר, מונחה תוכנת התקנה של שילובי ניסוי אופטימליים והגדלת עיצוב צעד חכמה. לכן, המתודולוגיה היא לא רק שימושית לאפיון ביטוי חלבון בצמחים, אלא גם לחקירה של מערכות מורכבות אחרות חסרות תיאור מכניסטי. משוואות החיזוי המתארות את הקישוריות בין פרמטרים ניתן להשתמש כדי להקים מודלים מכניסטית למערכות מורכבות אחרות.

מודל תיאורים עבור הצטברות DsRed בביטוי חולף באמצעות יזמים ו5&#39;UTRs שונים הקרינה DsRed בתמציות עלים שימשה כדי להצביע על רמת הביטוי של חלבון רקומביננטי וכך שימש כתגובה באסטרטגית DOE. הבדל גילוי המינימום שאנחנו נחשבים רלוונטיים ה...

Watch this Scientific Journal Video about Characterization of Complex Systems Using the Design of Experiments Approach: Transient Protein Expression in Tobacco as a Case Study at JoVE.com

Characterization of Complex Systems Using the Design of Experiments Approach: Transient Protein Expression in Tobacco as a Case Study

אנו מתארים עיצוב של גישת ניסויים שיכולים לשמש כדי לקבוע ולעצב את ההשפעה של אלמנטי transgene רגולציה, פרמטרים גדילה והתפתחות צמח, ותנאי דגירה על הביטוי החולף של נוגדנים חד שבטיים וחלבונים כתב בצמחים.

אפיון של מערכות מורכבות באמצעות העיצוב של גישת ניסויים: ביטוי חלבון חלוף בטבק כמקרה מבחן

Plants provide multiple benefits for the production of biopharmaceuticals including low costs, scalability, and safety. Transient expression offers the ...

characterization-complex-systems-using-design-experiments-approach

Research

JoVE Journal

Bioengineering

16.4K Views.  RWTH Aachen University.  אנו מתארים עיצוב של גישת ניסויים שיכולים לשמש כדי לקבוע ולעצב את ההשפעה של אלמנטי transgene רגולציה, פרמטרים גדילה והתפתחות צמח, ותנאי דגירה על הביטוי החולף של נוגדנים חד שבטיים וחלבונים כתב בצמחים. 

Video: Characterization of Complex Systems Using the Design of Experiments Approach: Transient Protein Expression in Tobacco as a Case Study

A Robotic Platform for High-throughput Protoplast Isolation and Transformation

Over the last decade there has been a resurgence in the use of plant protoplasts that range from model species to crop species, for analysis of signal transduction pathways, transcriptional regulatory networks, gene expression, genome-editing, and gene-silencing. Furthermore, significant progress has been made in the regeneration of plants from protoplasts, which has generated even more interest in the use of these systems for plant genomics. In this work, a protocol has been developed for automation of protoplast isolation and transformation from a &#39;Bright Yellow&#39; 2 (BY-2) tobacco suspension culture using a robotic platform. The transformation procedures were validated using an orange fluorescent protein (OFP) reporter gene (pporRFP) under the control of the Cauliflower mosaic virus 35S promoter (35S). OFP expression in protoplasts was confirmed by epifluorescence microscopy. Analyses also included protoplast production efficiency methods using propidium iodide. Finally, low-cost food-grade enzymes were used for the protoplast isolation procedure, circumventing the need for lab-grade enzymes that are cost-prohibitive in high-throughput automated protoplast isolation and analysis. Based on the protocol developed in this work, the complete procedure from protoplast isolation to transformation can be conducted in under 4 hr, without any input from the operator. While the protocol developed in this work was validated with the BY-2 cell culture, the procedures and methods should be translatable to any plant suspension culture/protoplast system, which should enable acceleration of crop genomics research.

A high-throughput, automated, tobacco protoplast production and transformation methodology is described. The robotic system enables massively parallel gene expression and discovery in the model BY-2 system that should be translatable to non-model crops.

פלטפורמה רובוטית עבור תפוקה גבוהה בידוד פרוטופלאסט וההשנאה

Over the last decade there has been a resurgence in the use of plant protoplasts that range from model species to crop species, for analysis of signal ...

Genetics

Transient Gene Expression in Tobacco using Gibson Assembly and the Gene Gun

In order to target a single protein to multiple subcellular organelles, plants typically duplicate the relevant genes, and express each gene separately using complex regulatory strategies including differential promoters and/or signal sequences. Metabolic engineers and synthetic biologists interested in targeting enzymes to a particular organelle are faced with a challenge: For a protein that is to be localized to more than one organelle, the engineer must clone the same gene multiple times. This work presents a solution to this strategy: harnessing alternative splicing of mRNA. This technology takes advantage of established chloroplast and peroxisome targeting sequences and combines them into a single mRNA that is alternatively spliced. Some splice variants are sent to the chloroplast, some to the peroxisome, and some to the cytosol. Here the system is designed for multiple-organelle targeting with alternative splicing. In this work, GFP was expected to be expressed in the chloroplast, cytosol, and peroxisome by a series of rationally designed 5&#8217; mRNA tags. These tags have the potential to reduce the amount of cloning required when heterologous genes need to be expressed in multiple subcellular organelles. The constructs were designed in previous work11, and were cloned using Gibson assembly, a ligation independent cloning method that does not require restriction enzymes. The resultant plasmids were introduced into Nicotiana benthamiana epidermal leaf cells with a modified Gene Gun protocol. Finally, transformed leaves were observed with confocal microscopy.

This work describes a novel method for selectively targeting subcellular organelles in plants, assayed using the BioRad Gene Gun.

ביטוי גנים חולף בטבק באמצעות גיבסון הרכבה והאקדח הגן

In order to target a single protein to multiple subcellular organelles, plants typically duplicate the relevant genes, and express each gene separately ...

Environment

Controllable Ion Channel Expression through Inducible Transient Transfection

Transfection, the delivery of foreign nucleic acids into a cell, is a powerful tool in protein research. Through this method, ion channels can be investigated through electrophysiological analysis, biochemical characterization, mutational studies, and their effects on cellular processes. Transient transfections offer a simple protocol in which the protein becomes available for analysis within a few hours to days. Although this method presents a relatively straightforward and time efficient protocol, one of the critical components is calibrating the expression of the gene of interest to physiological relevant levels or levels that are suitable for analysis. To this end, many different approaches that offer the ability to control the expression of the gene of interest have emerged. Several stable cell transfection protocols provide a way to permanently introduce a gene of interest into the cellular genome under the regulation of a tetracycline-controlled transcriptional activation. While this technique produces reliable expression levels, each gene of interest requires a few weeks of skilled work including calibration of a killing curve, selection of cell colonies, and overall more resources. Here we present a protocol that uses transient transfection of the Transient Receptor Potential cation channel subfamily V member 1 (TRPV1) gene in an inducible system as an efficient way to express a protein in a controlled manner which is essential in ion channel analysis. We demonstrate that using this technique, we are able to perform calcium imaging, whole cell, and single channel analysis with controlled channel levels required for each type of data collection with a single transfection. Overall, this provides a replicable technique that can be used to study ion channels structure and function.

Studying ion channels through a heterologously expressing system has become a core technique in biomedical research. In this manuscript, we present a time efficient method to achieve tightly controlled ion channel expression by performing transient transfection under the control of an inducible promoter.

ביטוי ערוץ יון לשליטה באמצעות Transfection ועין מתנהל חלוף

Transfection, the delivery of foreign nucleic acids into a cell, is a powerful tool in protein research. Through this method, ion channels can be ...

Transient Expression in Nicotiana Benthamiana Leaves for Triterpene Production at a Preparative Scale

The triterpenes are one of the largest and most structurally diverse families of plant natural products. Many triterpene derivatives have been shown to possess medicinally relevant biological activity. However, thus far this potential has not translated into a plethora of triterpene-derived drugs in the clinic. This is arguably (at least partially) a consequence of limited practical synthetic access to this class of compound, a problem that can stifle the exploration of structure-activity relationships and development of lead candidates by traditional medicinal chemistry workflows. Despite their immense diversity, triterpenes are all derived from a single linear precursor, 2,3-oxidosqualene. Transient heterologous expression of biosynthetic enzymes in N. benthamiana can divert endogenous supplies of 2,3-oxidosqualene towards the production of new high-value triterpene products that are not naturally produced by this host. Agro-infiltration is an efficient and simple means of achieving transient expression in N. benthamiana. The process involves infiltration of plant leaves with a suspension of Agrobacterium tumefaciens carrying the expression construct(s) of interest. Co-infiltration of an additional A. tumefaciens strain carrying an expression construct encoding an enzyme that boosts precursor supply significantly increases yields. After a period of five days, the infiltrated leaf material can be harvested and processed to extract and isolate the resulting triterpene product(s). This is a process that is linearly and reliably scalable, simply by increasing the number of plants used in the experiment. Herein is described a protocol for rapid preparative-scale production of triterpenes utilizing this plant-based platform. The protocol utilizes an easily replicable vacuum infiltration apparatus, which allows the simultaneous infiltration of up to four plants, enabling batch-wise infiltration of hundreds of plants in a short period of time.

Transient Expression in Nicotiana Benthamiana Leaves for Triterpene Production at a Preparative Scale

Transient heterologous expression of biosynthetic enzymes in Nicotiana benthamiana leaves can divert endogenous supplies of 2,3-oxidosqualene towards the production of new high-value triterpene products. Herein is described a detailed protocol for rapid (5 days) preparative-scale production of triterpenes and analogs utilizing this powerful plant-based platform.

ארעי ביטוי טבק Benthamiana משאיר לייצור טריטרפן בקנה מידה מפוח

The triterpenes are one of the largest and most structurally diverse families of plant natural products. Many triterpene derivatives have been shown to ...

Biochemistry

Efficient Agroinfiltration of Plants for High-level Transient Expression of Recombinant Proteins

Mammalian cell culture is the major platform for commercial production of human vaccines and therapeutic proteins. However, it cannot meet the increasing worldwide demand for pharmaceuticals due to its limited scalability and high cost. Plants have shown to be one of the most promising alternative pharmaceutical production platforms that are robust, scalable, low-cost and safe. The recent development of virus-based vectors has allowed rapid and high-level transient expression of recombinant proteins in plants. To further optimize the utility of the transient expression system, we demonstrate a simple, efficient and scalable methodology to introduce target-gene containing Agrobacterium into plant tissue in this study. Our results indicate that agroinfiltration with both syringe and vacuum methods have resulted in the efficient introduction of Agrobacterium into leaves and robust production of two fluorescent proteins; GFP and DsRed. Furthermore, we demonstrate the unique advantages offered by both methods. Syringe infiltration is simple and does not need expensive equipment. It also allows the flexibility to either infiltrate the entire leave with one target gene, or to introduce genes of multiple targets on one leaf. Thus, it can be used for laboratory scale expression of recombinant proteins as well as for comparing different proteins or vectors for yield or expression kinetics. The simplicity of syringe infiltration also suggests its utility in high school and college education for the subject of biotechnology. In contrast, vacuum infiltration is more robust and can be scaled-up for commercial manufacture of pharmaceutical proteins. It also offers the advantage of being able to agroinfiltrate plant species that are not amenable for syringe infiltration such as lettuce and Arabidopsis. Overall, the combination of syringe and vacuum agroinfiltration provides researchers and educators a simple, efficient, and robust methodology for transient protein expression. It will greatly facilitate the development of pharmaceutical proteins and promote science education.

Plants offer a novel system for the production of pharmaceutical proteins on a commercial scale that is more scalable, cost-efficient and safe than current expression paradigms. In this study, we report a simple and convenient, yet scalable approach to introduce target-gene containing Agrobacterium tumefaciens into plants for protein transient expression.

Agroinfiltration היעילה של צמחים לביטוי חולף ברמה גבוהה של חלבונים רקומביננטי

Mammalian cell culture is the major platform for commercial production of human vaccines and therapeutic proteins. However, it cannot meet the increasing ...

Biology

Optimization and Utilization of Agrobacterium-mediated Transient Protein Production in Nicotiana

Agrobacterium-mediated transient protein production in plants is a promising approach to produce vaccine antigens and therapeutic proteins within a short period of time. However, this technology is only just beginning to be applied to large-scale production as many technological obstacles to scale up are now being overcome. Here, we demonstrate a simple and reproducible method for industrial-scale transient protein production based on vacuum infiltration of Nicotiana plants with Agrobacteria carrying launch vectors. Optimization of Agrobacterium cultivation in AB medium allows direct dilution of the bacterial culture in Milli-Q water, simplifying the infiltration process. Among three tested species of Nicotiana, N. excelsiana (N. benthamiana &#215; N. excelsior) was selected as the most promising host due to the ease of infiltration, high level of reporter protein production, and about two-fold higher biomass production under controlled environmental conditions. Induction of Agrobacterium harboring pBID4-GFP (Tobacco mosaic virus-based) using chemicals such as acetosyringone and monosaccharide had no effect on the protein production level. Infiltrating plant under 50 to 100 mbar for 30 or 60 sec resulted in about 95% infiltration of plant leaf tissues. Infiltration with Agrobacterium laboratory strain GV3101 showed the highest protein production compared to Agrobacteria laboratory strains LBA4404 and C58C1 and wild-type Agrobacteria strains at6, at10, at77 and A4. Co-expression of a viral RNA silencing suppressor, p23 or p19, in N. benthamiana resulted in earlier accumulation and increased production (15-25%) of target protein (influenza virus hemagglutinin).

Optimization and Utilization of Agrobacterium-mediated Transient Protein Production in Nicotiana

Transient protein production in Nicotiana plants based on vacuum infiltration with Agrobacteria carrying launch vectors (Tobacco mosaic virus-based) is a rapid and economic approach to produce vaccine antigens and therapeutic proteins. We simplified the procedure and improved target accumulation by optimizing conditions of bacteria cultivation, selecting host species, and co-introducing RNA silencing suppressors.

אופטימיזציה וניצול<em&gt; Agrobacterium</emייצור חלבון חלוף&gt; בתיווך ב<em&gt; Nicotiana</em

Agrobacterium-mediated transient protein production in plants is a promising approach to produce vaccine antigens and therapeutic proteins within a short ...

Protein-protein Interactions Visualized by Bimolecular Fluorescence Complementation in Tobacco Protoplasts and Leaves

Many proteins interact transiently with other proteins or are integrated into multi-protein complexes to perform their biological function. Bimolecular fluorescence complementation (BiFC) is an in vivo method to monitor such interactions in plant cells. In the presented protocol the investigated candidate proteins are fused to complementary halves of fluorescent proteins and the respective constructs are introduced into plant cells via agrobacterium-mediated transformation. Subsequently, the proteins are transiently expressed in tobacco leaves and the restored fluorescent signals can be detected with a confocal laser scanning microscope in the intact cells. This allows not only visualization of the interaction itself, but also the subcellular localization of the protein complexes can be determined. For this purpose, marker genes containing a fluorescent tag can be coexpressed along with the BiFC constructs, thus visualizing cellular structures such as the endoplasmic reticulum, mitochondria, the Golgi apparatus or the plasma membrane. The fluorescent signal can be monitored either directly in epidermal leaf cells or in single protoplasts, which can be easily isolated from the transformed tobacco leaves. BiFC is ideally suited to study protein-protein interactions in their natural surroundings within the living cell. However, it has to be considered that the expression has to be driven by strong promoters and that the interaction partners are modified due to fusion of the relatively large fluorescence tags, which might interfere with the interaction mechanism. Nevertheless, BiFC is an excellent complementary approach to other commonly applied methods investigating protein-protein interactions, such as coimmunoprecipitation, in vitro pull-down assays or yeast-two-hybrid experiments.

Formation of protein complexes in vivo can be visualized by bimolecular fluorescence complementation. Interaction partners are fused to complementary parts of fluorescent tags and transiently expressed in tobacco leaves, resulting in a reconstituted fluorescent signal upon close proximity of the two proteins.

חלבונים אינטראקציות דמיינו ידי bimolecular הקרינה complementation בProtoplasts הטבק ועלים

Many proteins interact transiently with other proteins or are integrated into multi-protein complexes to perform their biological function. Bimolecular ...

A Comparative Analysis of Recombinant Protein Expression in Different Biofactories: Bacteria, Insect Cells and Plant Systems

Plant-based systems are considered a valuable platform for the production of recombinant proteins as a result of their well-documented potential for the flexible, low-cost production of high-quality, bioactive products.
In this study, we compared the expression of a target human recombinant protein in traditional fermenter-based cell cultures (bacterial and insect) with plant-based expression systems, both transient and stable.
For each platform, we described the set-up, optimization and length of the production process, the final product quality and the yields and we evaluated provisional production costs, specific for the selected target recombinant protein.
Overall, our results indicate that bacteria are unsuitable for the production of the target protein due to its accumulation within insoluble inclusion bodies. On the other hand, plant-based systems are versatile platforms that allow the production of the selected protein at lower-costs than Baculovirus/insect cell system. In particular, stable transgenic lines displayed the highest-yield of the final product and transient expressing plants the fastest process development. However, not all recombinant proteins may benefit from plant-based systems but the best production platform should be determined empirically with a case-by-case approach, as described here.

In this study the expression of a target human recombinant protein in different production platforms was compared. We focused on traditional fermenter-based cultures and on plants, describing the set-up of each system and highlighting, on the basis of the reported results, the inherent limits and advantages for each platform.

ניתוח השוואתי של ביטוי חלבון רקומביננטי בBiofactories שונה: חיידקים, תאי חרקים וצמחי מערכות

Plant-based systems are considered a valuable platform for the production of recombinant proteins as a result of their well-documented potential for the ...

Imaging Spatial Reorganization of a MAPK Signaling Pathway Using the Tobacco Transient Expression System

Visualization of dynamic signaling events in live cells has been a challenge. We expanded an established transient expression system, the biomolecular fluorescent complementation (BiFC) assay in tobacco epidermal cells, from testing protein-protein interaction to monitoring spatial distribution of signal transduction in plant cells. In this protocol, we used the BiFC assay to show that the interaction and the signaling between the Arabidopsis MAPKKK YODA and MAPK6 occur at the plasma membrane. When the scaffold protein BASL was co-expressed, the YODA-MAPK interaction redistributed and spatially co-polarized with CFP-BASL. This modified tobacco expression system allows for quick examination of signaling localization and dynamic changes (less than 4 days) and can accommodate multiple of fluorescent protein colors (at least three). We also presented detailed methods to quantify protein distribution (asymmetric spatial localization, or &#34;polarization&#34;) in tobacco cells. This advanced tobacco expression system has a potential to be used widely for quick testing of dynamic signaling events in live plant cells.

At the subcellular level, signaling events are dynamically modulated by developmental and environmental cues. Here we describe a protocol that employs the tobacco transient expression system to monitor dynamic protein-protein interaction and to disclose spatial organization of signal transduction in plant cells.

הדמית ארגון מחדש מרחבית של מסלול איתות MAPK שימוש במערכת הביטוי החולפת הטבק

Visualization of dynamic signaling events in live cells has been a challenge. We expanded an established transient expression system, the biomolecular ...

Transient Expression and Cellular Localization of Recombinant Proteins in Cultured Insect Cells

Heterologous protein expression systems are used for the production of recombinant proteins, the interpretation of cellular trafficking/localization, and the determination of the biochemical function of proteins at the sub-organismal level. Although baculovirus expression systems are increasingly used for protein production in numerous biotechnological, pharmaceutical, and industrial applications, nonlytic systems that do not involve viral infection have clear benefits but are often overlooked and underutilized. Here, we describe a method for generating nonlytic expression vectors and transient recombinant protein expression. This protocol allows for the efficient cellular localization of recombinant proteins and can be used to rapidly discern protein trafficking within the cell. We show the expression of four&#160;recombinant proteins in a commercially available insect cell line, including two aquaporin proteins from the insect Bemisia tabaci, as well as subcellular marker proteins specific for the cell plasma membrane and for intracellular lysosomes. All recombinant proteins were produced as chimeras with fluorescent protein markers at their carboxyl termini, which allows for the direct detection of the recombinant proteins. The double transfection of cells with plasmids harboring constructs for the genes of interest and a known subcellular marker allows for live cell imaging and improved validation of cellular protein localization.

Nonlytic insect cell expression systems are underutilized for production, cellular trafficking/localization, and recombinant protein functional analysis. Here, we describe methods to generate expression vectors and subsequent transient protein expression in commercially available lepidopteran cell lines. The co-localization of Bemisia tabaci aquaporins with subcellular fluorescent marker proteins is also presented.

ביטוי חולף ואת סלולר לוקליזציה של חלבונים רקומביננטי תאים בתרבית חרקים

Heterologous protein expression systems are used for the production of recombinant proteins, the interpretation of cellular trafficking/localization, and ...