I. ההפשרה תאים קח את הבקבוקון קפוא של TN5 תאים, להפשיר אותה אמבטיה במים 37 מעלות צלזיוס. הוסף 5 מ&quot;ל של מדיום אקספרס חרקים (Invitrogen, קטלוג מספר 10486) בבקבוק 2 25cm TC. שים לב אקספרס five מגיע ללא גלוטמין, צריך להוסיף 10 מ&quot;ל של סטרפטוקוקוס עט גלוטמין גלוטמין 100x או לבד עד 1 ליטר התקשורת. הוסף TN5 תאים לתוכו דגירה במשך 30 דקות ב 27 מעלות צלזיוס חממה. ואז, לשאוב את המדיום עם DMSO בעדינות מבלי להפריע תאים המצורפת בתחתית הבקבוק. הוסף 5 מ&quot;ל של מדיום טריים. השנייה. גידול והרחבת תאים צג התאים בכל יום. כאשר הבקבוק הוא כמעט 70% ומחוברות, להביא את התאים ההשעיה מדפיקות את הבקבוק משני הצדדים ולהעביר את התאים בקבוק 175cm 2 על ידי הוספת 21 מ&quot;ל של מדיום לבטא חרקים ו 5 מ&quot;ל של תרבית תאים. כל בקבוק T175 צריך מסביב בינוני 25-30 מ&quot;ל נפח סופי. פיצול בקבוק ומחוברות (כ 1000000 / ml קונצרט כלשהו.) 04/01 או 05/01 לפי הצורך. אל תתנו לגדול יותר תאים. ספירת הקטעים שיש לך לפצל את התאים. אל לצמוח לאורך קטע מספר 30 כי זה עלול לגרום להזדקנות של תאים והתוצאה תמוגה התא. כאשר אתם מגיעים בנפח הרצוי בריכוז של 1 מ&quot;ל / מיליון דולר, להדביק את התאים. אני בדרך כלל גדלים 2-2.5 ליטר, אשר בדרך כלל כמויות עד שבעים 175cm 2 צלוחיות וכ. 25 עד 30 מ&quot;ל בבקבוק / או חמש Erlenmeyer 1 ליטר צלוחיות וכ. 400ml ל -500 מ&quot;ל בבקבוק אחד הערה: אתה גם יכול לגדל תאים בקבוק T-175 תקע חותם או בקבוק קורנינג Erlenmeyer. התאים גדל Erlenmeyer צלוחיות הוא מהיר וקל יותר. ג. Titering וירוס על ידי שיטת נקודת הסיום דילול <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> 3000-5000 פלייט לכל התאים היטב לתחתית צלחת שטוחה 96 היטב 200ul של המדיום five SFM Express. בואו התאים להתיישב 27 מעלות צלזיוס למשך 30 דקות לפחות. </li><li style=";text-align:right;direction:rtl"> הפוך את דילול המניות הסידורי של baculovirus. אחד צריך לעשות 1 / 10 דילול בשורה אחת של צלחת 96 U היטב לתחתית בעזרת 250ul לכל טוב. דילולים מתקבלות בינוני חמישה SFM Express. </li><li style=";text-align:right;direction:rtl"> בעזרת טפטפת רב, להעביר סכום קבוע (בדרך כלל 20 ul) של דילולים שונים על התאים. </li><li style=";text-align:right;direction:rtl"> תרבות ב 27 ° C למשך 7 ימים. כדי למנוע אידוי של שורות לקצה, לעטוף את הקצוות של צלחת עם parafilm. </li><li style=";text-align:right;direction:rtl"> לאחר 7 ימים, לבדוק את הצלחת לקבוע אילו דילול של נגיף דלקת נותן 50% - במחצית בארות בריכוז זה וירוס נגועים). ואז להשתמש בנוסחה כדי לחשב את titer, המתבטא pfu / מ&quot;ל ​​(pfu-פלאק יחידות להרכיב). הנוסחה היא למצוא מדריכים baculovirus ביותר. </li></ol> IV. הדבקה של תאים חשוב מאוד לדעת את titer מדויק של הנגיף. הוסף את הכמות הנדרשת של הנגיף. להכנת מלאי ויראלי התאים צריך להיות נגוע על ריבוי של זיהום (משרד הפנים) של לא יותר מ 1 (1pfu כדי 1High חמש התא) גבוהה משרד הפנים מוביל הייצור של מוטציות ויראליות. עבור ייצור החלבון, הכמות הרגילה היא 50-10 MO1. דוגמה: כייל של baculovirus mCD1d = 1X108 pfu / ml הוסף הפנים = 1 (כדי להגביר את הנגיף) = 20X10 ^ 6 תאים / בקבוק / 1x10 ^ 8 pfu / ml = 200 UL / בקבוקון משרד הפנים = 5 עד 10 (לייצור חלבון) = 1ml כדי 2ml עבור 20x10 ^ 6 תאים / בקבוקון ביום 4 לאחר ההדבקה, מסתכל על תאים נגועים. הם צריכים להיות מנותקת, מרחפת, נקניקיות נפוחות יוצרים. V. Supernatants שחזור קחו את המדיום של צלוחיות נגוע, להעביר בקבוקי 250 מ&quot;ל כחול כובע פלקון ספינינג. הם יכולים להיות autoclaved ולעשות בהם שימוש חוזר. מלאו את הבקבוקים באופן שווה צנטריפוגות התאים Sorvall GSA הרוטור ב 200rpm, 20 דקות, 4 ° C. אסוף את supernatant והמשך לטיהור נוסף. VI. טיהור mCD1d רקומביננטי <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> דיאליזה וריכוז במאגר סודיום פוספט 150mm (pH 7.4) </li><li style=";text-align:right;direction:rtl"> על ידי Ni-agarose כרומטוגרפיה נ.ת. ע, elute החלבון mCD1d + b2m עם 150mm פוספט נתרן 200 mM Immidazole </li><li style=";text-align:right;direction:rtl"> הפעלה באמצעות טור Desalting 20mm טריס HCL pH8 חיץ להיפטר Immidazole </li><li style=";text-align:right;direction:rtl"> הפעל Exchange כרומטוגרפיה אניון באמצעות טור MonoQ לחסל את המזהמים הנותרים. </li><li style=";text-align:right;direction:rtl"> להתרכז כדי 1mg/ml באמצעות י&quot;מ 30 concentrator (Millipore) </li><li style=";text-align:right;direction:rtl"> Biotynlation בירה האנזימטית של mCD1d על פי פרוטוקול של היצרן (רעבתנות) </li><li style=";text-align:right;direction:rtl"> ביוטין חינם מסולק על ידי טור S200 (הרחקה Chromatograpy גודל) באמצעות בופר פוספט (PBS), pH7.4. ממוצע של 2 עד 3 מקלעים של חלבון biotynlated ניתן לקבל 2 ליטר של supernatant </li><li style=";text-align:right;direction:rtl"> הפקה של-Galcer CD1d tetramer. </li></ol> על מנת לטעון מולקולה CD1d עם-Galcer, מסיסים biotynlated CD1d-B2מ &#39;הוא מודגרות לילה עם עודף three טוחנת קיפול של Galcer-a, מומס 0.5% ב -20 Tween PBS, כלוריד הנתרן O.9% בטמפרטורת החדר. Tetramers נוצרים על ידי ערבוב של-Galcer מונומרים טעון CD1d עם עודף של פי 4 טוחנת PE - streptavidin מצומדות ו דוגרים במשך 4 שעות בטמפרטורת החדר. חנות ב 48 ° C.

<ol>
	<li>Benlagha, K., Weiss, A., Beavis, A., Teyton, L., Bendelac, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+identification+of+glycolipipd+antigen-specific+T+cells+using+fluorecent+CD1d+tetramers.">In vivo identification of glycolipipd antigen-specific T cells using fluorecent CD1d tetramers.</a> J. Exp. Med. 191, 1895-1903 (2000).</li><li>Kinjo, Y., Wu, D., Kim, G., Xing, G. W., Poles, M. A., Ho, D. D., Tsuji, M., Kawahara, K., Wong, C. H., Kronenberg, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recognition+of+bacterial+glycosphingolipids+by+natural+killer+T+cells.">Recognition of bacterial glycosphingolipids by natural killer T cells.</a> Nature. 434, 520-525 (2005).</li><li>Naidenko, O. V., Maher, J. K., Ernst, W. A., Sakai, T., Modlin, R. L., Kronenberg, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Binding+and+antigen+presentation+of+ceramide-containing+glycolipids+by+soluble+mouse+and+human+CD1d+molecules.">Binding and antigen presentation of ceramide-containing glycolipids by soluble mouse and human CD1d molecules.</a> J. Exp. Med. 190, 1069-1080 (1999).</li><li>Porcelli, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+CD1+family:+A+third+lineage+of+antigen-+presenting+molecules.">The CD1 family: A third lineage of antigen- presenting molecules.</a> Adv. Immunol. 59, 1-98 (1995).</li><li>Sidobre, S., Kronemberg, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CD1+tetramers:+A+powerful+tool+for+the+analysis+of+glycolipid-reactive+T+cells.">CD1 tetramers: A powerful tool for the analysis of glycolipid-reactive T cells.</a> J. Immunol. 169, 1340-1348 (2002).</li></ol>

בהליך זה, הוא הראה כיצד ליזום, לגדול, להדביק תאים חרקים וכיצד titer baculovirus באמצעות תאים אלה. ההיבטים החשובים ביותר של הליך זה הם אחזקה של תאים לדעת את titer המדויק של baculovirus. ברגע שיש לך שנוצר CD1 tetramers, הם יכולים לשמש כלי רב עוצמה לניתוח glycolipid בתאי T תגובתי. מערכות Baculovirus משמשים גם נרחב כדי ...

<!DOCTYPE html PUBLIC "-//W3C//DTD XHTML 1.0 Transitional//EN" "http://www.w3.org/TR/xhtml1/DTD/xhtml1-transitional.dtd">
<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
		
		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Express Five SFM</td>
<td> Serum Free Medium</td>
<td>Invitrogen</td>
<td>10486025</td>
<td>&nbsp;</td>
<tr>
<td>High Five TM</td>
<td> Insect Cells</td>
<td>Invitrogen</td>
<td>B855-02</td>
<td>&nbsp;</td>
<tr>
<td>Glutamine Pen strep</td>
<td>Antibiotics</td>
<td>Invitrogen</td>
<td>10378-016</td>
<td>&nbsp;</td>
<tr>
<td>25cm2 TC Flask</td>
<td> Plastic</td>
<td>Fisher</td>
<td>10-126-28</td>
<td>&nbsp;</td>
<tr>
<td>175 cm2 TC Flask</td>
<td> Plug seal flask</td>
<td>Fisher</td>
<td>10-126-8</td>
<td>&nbsp;</td>
<tr>
<td>Erlenmeyer Flask</td>
<td> Cell Culture Flask</td>
<td>Fisher</td>
<td>07 200 672</td>
<td>&nbsp;</td>
<tr>
<td>YM 30 Concenterator</td>
<td>30,000 MWCO</td>
<td>Millipore</td>
<td>UFC 903096</td>
<td>&nbsp;</td>
<tr>
<td>FPLC equipment</td>
<td>&nbsp;</td>
<td>GE Healthcare</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>BrA Enzyme and Buffer</td>
<td> Biotinylation Kit</td>
<td>Avidity</td>
<td>BIRA500</td>
<td>&nbsp;</td>
<tr>
<td>Ni-NTA Agarose Column</td>
<td>His Tag Protein Purification Column</td>
<td>GE Healthcare</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Desalting column</td>
<td> To get Rod of Salt from Protein</td>
<td>GE Healthcare</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>MonoQ Column</td>
<td>Anion Exchange Chromatgraphy</td>
<td>GE Healthcare</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>S200 Column</td>
<td> Size Exclusion Chromatography</td>
<td>GE Healthcare</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>alpha-Galcer</td>
<td> Glycolipid</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>PE- Streptavidin</td>
<td> For conjugating Protein</td>
<td>Molecular Probe</td>
<td> S-866</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

a method for production of recombinant mcd1d protein in insect cells.

CD1 חלבונים מהווים המחזור השלישי של אנטיגן הצגת מולקולות. הם פני התא גליקופרוטאינים, כפי שבאה לידי ביטוי שרשראות כבדות כ 50-kDa glycosylated המשויכים noncovalently עם microglobulin-Beta2. הם נקשרים שומנים ולא פפטידים. למרות המבנה שלהם מאשר את הדמיון של CD1 חלבונים MHC אני בכיתה ו הכיתה מולקולות II הצגת אנטיגן, mCD1d חריץ צר יחסית, עמוק, הידרופובי מאוד ויש לו שני כיסים מחייב במקום הכיסים רדוד מספר תיאר הקלאסית MHC- מקודד אנטיגן הצגת מולקולות. בהתבסס על רצפי אמינו חומצה שלהם, כגון חריץ hydrobphobic מספקת סביבה אידיאלית עבור הכריכה של אנטיגנים השומנים. הרוצח טבעי T (NKT) תאים להשתמש TCR שלהם להכיר glycolipids חייב או שהציג CD1d. בתאי T תגובתי שומנים שהציג CD1 היו מעורבים הגנה נגד מחלות אוטואימוניות זיהומיות בבית דחייה הגידול. לפיכך, היכולת לזהות, לטהר, ולעקוב אחר תגובת התא NKT CD1-reactive היא בעלת חשיבות רבה. הדור של tetramers של אלפא Galactosyl ceramide (א-Galcer) עם CD1d יש תובנה משמעותית לתוך הביולוגיה של תאים NKT. Tetramers בנויים CD1 מולקולות אחרות יש גם שנוצר אלה ריאגנטים חדשים הרחיבו מאוד את הידע של הפונקציות של שומנים בדם תגובתי בתאי T, עם שימוש פוטנציאלי ניטור התגובה השומנים מבוסס חיסונים באבחון של מחלות אוטואימוניות אחרות טיפולים.

Watch this Scientific Journal Video about A Method For Production of Recombinant mCD1d Protein in Insect Cells. at JoVE.com

A Method For Production of Recombinant mCD1d Protein in Insect Cells.

שיטה להכנת תאים חרקים להדביק אותם עם baculovirus לצורך הייצור של proteinand רקומביננטי mCD1d יצירת tetramers mCD1d.

שיטה לייצור של חלבון רקומביננטי mCD1d בתוך תאים חרקים.

CD1 proteins constitute a third class of antigen-presenting molecules. They are cell surface glycoproteins, expressed as approximately 50-kDa glycosylated ...

a-method-for-production-of-recombinant-mcd1d-protein-in-insect-cells

Research

JoVE Journal

Biology

10.7K Views.  La Jolla Institute for Allergy and Immunology. שיטה להכנת תאים חרקים להדביק אותם עם baculovirus לצורך הייצור של proteinand רקומביננטי mCD1d יצירת tetramers mCD1d.

Video: A Method For Production of Recombinant mCD1d Protein in Insect Cells.

Pyrosequencing: A Simple Method for Accurate Genotyping

Pharmacogenetic research benefits first-hand from the abundance of information provided by the completion of the Human Genome Project. With such a tremendous amount of data available comes an explosion of genotyping methods. Pyrosequencing(R) is one of the most thorough yet simple methods to date used to analyze polymorphisms. It also has the ability to identify tri-allelic, indels, short-repeat polymorphisms, along with determining allele percentages for methylation or pooled sample assessment. In addition, there is a standardized control sequence that provides internal quality control. This method has led to rapid and efficient single-nucleotide polymorphism evaluation including many clinically relevant polymorphisms. The technique and methodology of Pyrosequencing is explained.


Pyrosequencing(R) is one of the most thorough yet simple methods to date used to analyze polymorphisms. This method has led to rapid and efficient single-nucleotide polymorphism evaluation including many clinically relevant polymorphisms. The technique and methodology of Pyrosequencing is explained.

Pyrosequencing: שיטה פשוטה genotyping מדויק

Pharmacogenetic research benefits first-hand from the abundance of information provided by the completion of the Human Genome Project. With such a ...

A Protocol for Phage Display and Affinity Selection Using Recombinant Protein Baits

Using recombinant phage as a scaffold to present various protein portions encoded by a directionally cloned cDNA library to immobilized bait molecules is an efficient means to discover interactions. The technique has largely been used to discover protein-protein interactions but the bait molecule to be challenged need not be restricted to proteins. The protocol presented here has been optimized to allow a modest number of baits to be screened in replicates to maximize the identification of independent clones presenting the same protein. This permits greater confidence that interacting proteins identified are legitimate interactors of the bait molecule. Monitoring the phage titer after each affinity selection round provides information on how the affinity selection is progressing as well as on the efficacy of negative controls. One means of titering the phage, and how and what to prepare in advance to allow this process to progress as efficiently as possible, is presented. Attributes of amplicons retrieved following isolation of independent plaque are highlighted that can be used to ascertain how well the affinity selection has progressed. Trouble shooting techniques to minimize false positives or to bypass persistently recovered phage are explained. Means of reducing viral contamination flare up are discussed.

Phage display is a powerful technique to capture proteins or protein moieties that interact with an immobilized molecule of interest. Once a decision of the type of phage cDNA library to create and screen has been made, the protocol described here permits efficient affinity selection leading to identification of interactors.

פרוטוקול לתצוגת הפאג וזיקה בחירה באמצעות פיתיונות חלבון רקומביננטי

Using  recombinant phage as a scaffold to present various protein portions encoded by  a directionally cloned cDNA library to immobilized bait molecules ...

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 ...

Efficient Production and Purification of Recombinant Murine Kindlin-3 from Insect Cells for Biophysical Studies

Kindlins are essential coactivators, with talin, of the cell surface receptors&#160;integrins and also participate in integrin outside-in signalling, and the control of gene transcription in the cell nucleus. The kindlins are ~75 kDa multidomain proteins and bind to an NPxY motif and upstream T/S cluster of the integrin &#946;-subunit cytoplasmic tail. The hematopoietically-important kindlin isoform, kindlin-3, is critical for platelet aggregation during thrombus formation, leukocyte rolling in response to infection and inflammation and osteoclast podocyte formation in bone resorption. Kindlin-3&#39;s role in these processes has resulted in extensive cellular and physiological studies. However, there is a need for an efficient method of acquiring high quality milligram quantities of the protein for further studies. We have developed a protocol, here described, for the efficient expression and purification of recombinant murine kindlin-3 by use of a baculovirus-driven expression system in Sf9 cells yielding sufficient amounts of high purity full-length protein to allow its biophysical characterization. The same approach could be taken in the study of the other mammalian kindlin isoforms.

Kindlins are fundamental to cell adhesion through integrins but studies of them have been hampered by the difficulty encountered in expressing them recombinantly in bacterial hosts. We describe here methods for their efficient production in baculovirus-infected insect cells.

הפקה וטיהור יעילה של רקומביננטי Murine Kindlin-3 מתאי חרקים לBiophysical לימודים

Kindlins are essential coactivators, with talin, of the cell surface receptors&#160;integrins and also participate in integrin outside-in signalling, and ...

Investigating Protein-protein Interactions in Live Cells Using Bioluminescence Resonance Energy Transfer

Assays based on Bioluminescence Resonance Energy Transfer (BRET) provide a sensitive and reliable means to monitor protein-protein interactions in live cells. BRET is the non-radiative transfer of energy from a 'donor' luciferase enzyme to an 'acceptor' fluorescent protein. In the most common configuration of this assay, the donor is Renilla reniformis luciferase and the acceptor is Yellow Fluorescent Protein (YFP). Because the efficiency of energy transfer is strongly distance-dependent, observation of the BRET phenomenon requires that the donor and acceptor be in close proximity. To test for an interaction between two proteins of interest in cultured mammalian cells, one protein is expressed as a fusion with luciferase and the second as a fusion with YFP. An interaction between the two proteins of interest may bring the donor and acceptor sufficiently close for energy transfer to occur. Compared to other techniques for investigating protein-protein interactions, the BRET assay is sensitive, requires little hands-on time and few reagents, and is able to detect interactions which are weak, transient, or dependent on the biochemical environment found within a live cell. It is therefore an ideal approach for confirming putative interactions suggested by yeast two-hybrid or mass spectrometry proteomics studies, and in addition it is well-suited for mapping interacting regions, assessing the effect of post-translational modifications on protein-protein interactions, and evaluating the impact of mutations identified in patient DNA.

Interactions between proteins are fundamental to all cellular processes. Using Bioluminescence Resonance Energy Transfer, the interaction between a pair of proteins can be monitored in live cells and in real time. Furthermore, the effects of potentially pathogenic mutations can be assessed.

חוקר חלבונים אינטראקציות בתאים חיים באמצעות העברת פליטת אור התהודה אנרגיה

Assays based on Bioluminescence Resonance Energy Transfer (BRET) provide a sensitive and reliable means to monitor protein-protein interactions in live ...

Recombinant Protein Expression for Structural Biology in HEK 293F Suspension Cells: A Novel and Accessible Approach

The expression and purification of large amounts of recombinant protein complexes is an essential requirement for structural biology studies. For over two decades, prokaryotic expression systems such as E. coli have dominated the scientific literature over costly and less efficient eukaryotic cell lines. Despite the clear advantage in terms of yields and costs of expressing recombinant proteins in bacteria, the absence of specific co-factors, chaperones and post-translational modifications may cause loss of function, mis-folding and can disrupt protein-protein interactions of certain eukaryotic multi-subunit complexes, surface receptors and secreted proteins. The use of mammalian cell expression systems can address these drawbacks since they provide a eukaryotic expression environment. However, low protein yields and high costs of such methods have until recently limited their use for structural biology. Here we describe a simple and accessible method for expressing and purifying milligram quantities of protein by performing transient transfections of suspension grown HEK (Human Embryonic Kidney) 293F cells.

The expression of recombinant proteins by mammalian systems is becoming an attractive method for producing protein complexes for structural biology. Here we present a simple yet highly efficient expression system using suspension grown mammalian cells to purify protein complexes for structural studies.

ביטוי חלבון רקומביננטי לביולוגיה מבנית בתאי ההשעיה HEK 293F: רומן וגישה נגישה

The expression and purification of large amounts of recombinant protein complexes is an essential requirement for structural biology studies. For over two ...

Green Fluorescent Protein-based Expression Screening of Membrane Proteins in Escherichia coli

The production of recombinant membrane proteins for structural and functional studies remains technically challenging due to low levels of expression and the inherent instability of many membrane proteins once solubilized in detergents. A protocol is described that combines ligation independent cloning of membrane proteins as GFP fusions with expression in Escherichia coli detected by GFP fluorescence. This enables the construction and expression screening of multiple membrane protein/variants to identify candidates suitable for further investment of time and effort. The GFP reporter is used in a primary screen of expression by visualizing GFP fluorescence following SDS polyacrylamide gel electrophoresis (SDS-PAGE). Membrane proteins that show both a high expression level with minimum degradation as indicated by the absence of free GFP, are selected for a secondary screen. These constructs are scaled and a total membrane fraction prepared and solubilized in four different detergents. Following ultracentrifugation to remove detergent-insoluble material, lysates are analyzed by fluorescence detection size exclusion chromatography (FSEC). Monitoring the size exclusion profile by GFP fluorescence provides information about the mono-dispersity and integrity of the membrane proteins in different detergents. Protein: detergent combinations that elute with a symmetrical peak with little or no free GFP and minimum aggregation are candidates for subsequent purification. Using the above methodology, the heterologous expression in E. coli of SED (shape, elongation, division, and sporulation) proteins from 47 different species of bacteria was analyzed. These proteins typically have ten transmembrane domains and are essential for cell division. The results show that the production of the SEDs orthologues in E. coli was highly variable with respect to the expression levels and integrity of the GFP fusion proteins. The experiment identified a subset for further investigation.

Green Fluorescent Protein-based Expression Screening of Membrane Proteins in Escherichia coli

A streamlined approach to screening for the expression of recombinant membrane proteins in Escherichia coli based on fusion to green fluorescent protein is presented.

הקרנה ירוקה פלורסנט מבוסס חלבון ביטוי של החלבונים ממברנה ב<em&gt; Coli Escherichia</em

The production of recombinant membrane proteins for structural and functional studies remains technically challenging due to low levels of expression and ...

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 ...

Nephrotoxin Microinjection in Zebrafish to Model Acute Kidney Injury

The kidneys are susceptible to harm from exposure to chemicals they filter from the bloodstream. This can lead to organ injury associated with a rapid decline in renal function and development of the clinical syndrome known as acute kidney injury (AKI). Pharmacological agents used to treat medical circumstances ranging from bacterial infection to cancer, when administered individually or in combination with other drugs, can initiate AKI. Zebrafish are a useful animal model to study the chemical effects on renal function in vivo, as they form an embryonic kidney comprised of nephron functional units that are conserved with higher vertebrates, including humans. Further, zebrafish can be utilized to perform genetic and chemical screens, which provide opportunities to elucidate the cellular and molecular facets of AKI and develop therapeutic strategies such as the identification of nephroprotective molecules. Here, we demonstrate how microinjection into the zebrafish embryo can be utilized as a paradigm for nephrotoxin studies.

Renal injuries incurred from nephrotoxins, which include drugs ranging from antibiotics to chemotherapeutics, can result in complex disorders whose pathogenesis remains incompletely understood. This protocol demonstrates how zebrafish can be used for disease modeling of these conditions, which can be applied to the identification of renoprotective measures.

Nephrotoxin Microinjection דג הזברה למודל אי-ספיקת כליות חריפה

The kidneys are susceptible to harm from exposure to chemicals they filter from the bloodstream. This can lead to organ injury associated with a rapid ...

A Graphical User Interface for Software-assisted Tracking of Protein Concentration in Dynamic Cellular Protrusions

Filopodia are dynamic, finger-like cellular protrusions associated with migration and cell-cell communication. In order to better understand the complex signaling mechanisms underlying filopodial initiation, elongation and subsequent stabilization or retraction, it is crucial to determine the spatio-temporal protein activity in these dynamic structures. To analyze protein function in filopodia, we recently developed a semi-automated tracking algorithm that adapts to filopodial shape-changes, thus allowing parallel analysis of protrusion dynamics and relative protein concentration along the whole filopodial length. Here, we present a detailed step-by-step protocol for optimized cell handling, image acquisition and software analysis. We further provide instructions for the use of optional features during image analysis and data representation, as well as troubleshooting guidelines for all critical steps along the way. Finally, we also include a comparison of the described image analysis software with other programs available for filopodia quantification. Together, the presented protocol provides a framework for accurate analysis of protein dynamics in filopodial protrusions using image analysis software.

We present a software solution for semi-automated tracking of relative protein concentration along the length of dynamic cellular protrusions.

ממשק משתמש גרפי למעקב בסיוע תוכנה של ריכוז חלבון בהפרעות ניידות דינמיות

Filopodia are dynamic, finger-like cellular protrusions associated with migration and cell-cell communication. In order to better understand the complex ...

שיטה לייצור של חלבון רקומביננטי mCD1d בתוך תאים חרקים.

Summary

Explore More Videos

Chapters in this video