עבודה זו נתמכה על ידי HIV רשת אונטריו טיפול מחקר הפעלה גרנט (ROG-G645) וקנדה מכון פרס מחקר לחוקר הבריאות החדש (MSH-113554) כדי JEL, ואוניברסיטת טורונטו מלגות חה, FCA, וג&#39;וינט. המחברים מבקשים להודות מארני Fusco, דפנה אבלסון וד&quot;ר אריקה Ollmann Saphire במכון המחקר סקריפס (La Jolla, CA) עבור תאים המספקים, ebolavirus וקטור GP ביטוי וייעוץ כללי.

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אין ניגוד עניינים הצהיר.

את 10-שכבת מפעלים סלולריים הם כלי יעיל לייצור כמויות מיליגרם של חלבון. היתרון הגדול של שימוש מפעל התא על כלי מסורתיים אחרים, כגון בקבוקים, צלוחיות הרים לוחצים או צלוחיות תיבת הטווח, היא שהם אינם דורשים רכישת ציוד מעבדה נוספת. תקן ה-CO 2 באינקובטור (~~~HEAD=NNS 6.0 מ&quot;ק. מט...

<table border="1" style=";text-align:right;direction:rtl"><tbody><tr><td> שם מגיב </td><td> חברה </td><td> מספר קטלוגי </td><td> תגובות </td></tr><tr><td> אלקליין phosphatase (BCIP / NBT) מצע נוזלי פתרון </td><td> סיגמא </td><td> B6404 </td><td></td></tr><tr><td> אנטיביוטיקה / Antimycotic, 100x </td><td> Invitrogen </td><td> 15240062 </td><td></td></tr><tr><td> Anti-HA זיקה מטריקס, שיבוט 3F10 </td><td> רוש </td><td> 1815016 </td><td></td></tr><tr><td> Anti-HA Murine MAB, שיבוט 16B12 </td><td> Covance </td><td> MMS-101P </td><td></td></tr><tr><td> תרבות בקבוק נייד, T75 2 ס&quot;מ תרבות רקמות טיפול </td><td> קורנינג </td><td> 430641 </td><td></td></tr><tr><td> תרבות בקבוק נייד, T225 2 ס&quot;מ תרבות רקמות טיפול </td><td> קורנינג </td><td> 431082 </td><td></td></tr><tr><td>תרבות צלחות סלולריים ,6-גם תרביות רקמה טיפול </td><td> קורנינג </td><td> 3516 </td><td></td></tr><tr><td> מפעל נייד, 10-שכבת CellSTACK </td><td> קורנינג </td><td> 3312 </td><td></td></tr><tr><td> Centramate אומגה 5K קלטת </td><td> אריג אבל </td><td> OS005C12 </td><td></td></tr><tr><td> Centramate אומגה 30K קלטת </td><td> אריג אבל </td><td> OS030C12 </td><td></td></tr><tr><td> עמודות כרומטוגרפיה זכוכית, 1.0x10 ס&quot;מ </td><td> Kontes </td><td> 4204001010 </td><td></td></tr><tr><td> ציפרופלוקסאצין </td><td> סיגמא </td><td> 17850 </td><td></td></tr><tr><td> CO 2 </td><td></td><td></td><td></td></tr><tr><td> שונה Dulbecco של הנשר של התקשורת (DMEM) </td><td> סיגמא </td><td> D5796 </td><td></td></tr><tr><td> בסרום שור עוברית (FBS), חום מומת </td><td> Invitrogen </td><td> 12484-028 </td><td></td></tr><tr><td> FuGENE HD מגיב transfection </td><td> Promega </td><td> 4709691001 </td><td></td></tr><tr><td> GeneJuice מגיב transfection </td><td> EMD / מרק </td><td> 70967-6 </td><td></td></tr><tr><td> גליצין </td><td> סיגמא </td><td> G8898 </td><td></td></tr><tr><td> נגד העכבר עיזים IgG ו &#39;(&quot;א.ב.) 2 אלקליין </td><td> Thermo Scientific </td><td> 31324 </td><td></td></tr><tr><td> phosphatase-מצומדות נוגדן </td><td></td><td></td><td></td></tr><tr><td> Hemagglutinin (HA) פפטיד, 100 מ&quot;ג </td><td> Genscript </td><td> אישית סינתזה </td><td></td></tr><tr><td> (רצף: YPYDVPDYA, טוהר 95%) </td><td></td><td></td><td></td></tr><tr><td> HEK 293T תאים </td><td> ATCC </td><td> CRL-11268 </td><td></td></tr><tr><td> Householד אקונומיקה (4% w / v hypochlorite נתרן) </td><td> מותגים שונים זמינים </td><td></td><td></td></tr><tr><td> Immobilon-P PVDF הממברנה </td><td> Millipore </td><td> IPVH07850 </td><td></td></tr><tr><td> MiniPrep ערכת טיהור פלסמיד, PureLink מהירה </td><td> Invitrogen </td><td> K2100-11 </td><td></td></tr><tr><td> MaxiPrep ערכת טיהור פלסמיד, PureLink HiPure </td><td> Invitrogen </td><td> K2100-07 </td><td></td></tr><tr><td> 3 NaN </td><td> סיגמא </td><td> S8032 </td><td></td></tr><tr><td> הביטוי pDISPLAY וקטור </td><td> Invitrogen </td><td> V660-20 </td><td></td></tr><tr><td> פניצילין / סטרפטומיצין (עט / דלקת), 100x </td><td> Invitrogen </td><td> 15140-122 </td><td></td></tr><tr><td> פוספט בופר סליין (PBS), סטרילית 1X </td><td> סיגמא </td><td> D8537 </td><td></td></tr><tr><td> Polyethyleneimine (PEI), ליניארי 25 kDa </td><td> Polyscience </td><td> 23966 </td><td></td></tr><tr><td> אבקת חלב רזה יבש </td><td> צפורן </td><td></td><td></td></tr><tr><td> Stericup-GP ואקום PES סינון יחידה, </td><td> Millipore </td><td> SCGPU05RE </td><td></td></tr><tr><td> 0.22 מיקרומטר, 500 מ&quot;ל קיבולת </td><td></td><td></td><td></td></tr><tr><td> Trypan כחול </td><td> Invitrogen </td><td> 15250061 </td><td></td></tr><tr><td> טריפסין, EDTA, 0.05% (w / v) </td><td> Invitrogen </td><td> 25300-054 </td><td></td></tr><tr><td> Tween-20 </td><td> סיגמא </td><td> P7949 </td><td></td></tr><tr><td> חומצה ולפרואית </td><td> סיגמא </td><td> P4543 </td><td></td></tr><tr><td> Centramate מערכת זרימת משיק </td><td> אריג אבל </td><td></td><td></td></tr><tr><td> CO 2 באינקובטור humidified, תקן 6.0 מ&quot;ק. מטר </td><td> מותגים שונים זמינים </td><td></td><td></td></tr><tr><td> אלקטרופורזה והעברת היחידה </td><td> מותגים שונים זמינים </td><td></td><td></td></tr><tr><td> החממה, 37 ° C </td><td> מותגים שונים זמינים </td><td></td><td></td></tr></tbody></table>

a convenient and general expression platform for the production of secreted proteins from human cells

רקומביננטי ביטוי החלבון חיידקים, בדרך כלל א &#39; coli, הייתה האסטרטגיה המוצלחת ביותר עבור ביטוי בכמות מיליגרם של חלבונים. עם זאת, המארחים פרוקריוטים הם לעתים קרובות לא בהתאם ביטוי של חלבונים אנושיים, ויראלי או האיקריוטים בשל הרעילות של מקרומולקולה החוץ, ההבדלים מכונות קיפול חלבונים, או בשל חוסר שיתוף שינויים או שלאחר translational מסוימים של חיידקים. מערכות ההבעה על בסיס שמרים (pastoris פ או cerevisiae ס) 1,2, baculovirus-נגוע חרק (ס &#39;frugiperda או ט&#39; ני) 3 תאים, תא ללא במבחנה תרגום מערכות 2,4 שימשו בהצלחה לייצר חלבונים יונקים. באופן אינטואיטיבי, את ההתאמה הטובה ביותר היא להשתמש המארח יונקים על מנת להבטיח את הייצור של חלבונים רקומביננטיים המכילים הנכון לאחר שינויים של תרגום. מספר שורות תאים יונקים (קיד עובריים אנושייםניי (HEK) 293, C-V-1 התאים O rigin הנושאים V40 S larget T-antigen (COS), השחלות אוגר סיני (CHO), ועוד) כבר נוצלו בהצלחה overexpress כמויות מיליגרם של מספר חלבונים אנושיים 5-9. עם זאת, היתרונות של שימוש בתאי יונקים הם לעתים קרובות הגיב עלויות גבוהות יותר, הדרישה של ציוד מעבדה מיוחד, תשואות נמוכות יותר חלבון, ושעות ארוכות לפתח יציבות התא קמטי הבעה. הגדלת התשואה חלבונים לייצר מהר יותר, תוך שמירה על עלויות נמוכות, הם הגורמים העיקריים למעבדות אקדמיים ומסחריים רבים. כאן אנו מתארים זמן ועלות יעיל, חלק 2, הליך לביטוי של חלבונים המופרשים אדם חסיד תאים HEK 293T. מערכת זו הוא מסוגל לייצר מיקרוגרם כמויות מיליגרם של חלבון פונקציונלי ללימודי מבניים, biophysical ו ביוכימי. החלק הראשון, בונה רבות של הגן של עניין הם מייצרים<html> ד במקביל transfected זמני אל חסיד תאים HEK 293T בקנה מידה קטן. זיהוי וניתוח של חלבון רקומביננטי מופרש לתוך המדיום תרבית תאים מבוצע על ידי ניתוח כתם המערבי באמצעות נוגדנים זמינים מסחרית המכוונים נגד תג וקטור המקודד טיהור חלבונים. לאחר מכן, בונה מתאים לייצור בקנה מידה גדול חלבון transfected זמני באמצעות polyethyleneimine (PEI) ב -10 מפעלים שכבת תאים. חלבונים המופרשים לתוך ליטר למוצרי כרכים של המדיום אוויר מרוכזים לתוך כמויות לניהול באמצעות סינון זרימת משיק, ואחריו טיהור ידי כרומטוגרפיה נגד HA זיקה. השירות של פלטפורמה זו היא הוכחה על ידי היכולת שלו לבטא את כמויות מיליגרם של ציטוקינים, קולטנים ציטוקינים, קולטנים בתא השטח, גורמים פנימיים, וכן הגבלה גליקופרוטאינים ויראליים. בשיטה זו השתמשו בהצלחה גם בקביעת המבנה של 5,10 גליקופרוטאין trimeric ebolavirus. ילדה = &quot;jove_content&quot;&gt; לסיכום, פלטפורמה זו מציעה מהירות וקלות שימוש, ויכולת הרחבה תוך מיקסום איכות החלבון ופונקציונליות. יתר על כן, הציוד ללא תוספת, למעט תקן humidified CO 2 באינקובטור, נדרשת. הליך זה עשוי להתרחב במהירות למערכות מורכבות יותר, כמו ביטוי משותף של קומפלקסים חלבונים, אנטיגנים ונוגדנים, ייצור של וירוסים כמו חלקיקים עבור חיסונים, או לייצור של adenoviruses או lentiviruses עבור התמרה של שורות תאים קשים.

Watch this Scientific Journal Video about A Convenient and General Expression Platform for the Production of Secreted Proteins from Human Cells at JoVE.com

A Convenient and General Expression Platform for the Production of Secreted Proteins from Human Cells

בעידן שאחרי האדם הגנומיקה, הזמינות של חלבונים רקומביננטיים של תצורות המקומיים הוא חיוני למחקר מבנית, תפקודית טיפולית ופיתוח. כאן אנו מתארים מבחן רחב היקף של ביטוי חלבונים מערכת עובריים אנושיים לתאי כליה 293T, שניתן להשתמש בהם כדי לייצר מגוון של חלבונים רקומביננטיים.

פלטפורמת ביטוי נוח כללי לייצור חלבונים המופרשים מ תאים אנושיים

Recombinant protein expression in bacteria, typically E. coli, has been the most successful strategy for milligram quantity expression of proteins. ...

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21.0K Views.  University of Toronto. בעידן שאחרי האדם הגנומיקה, הזמינות של חלבונים רקומביננטיים של תצורות המקומיים הוא חיוני למחקר מבנית, תפקודית טיפולית ופיתוח. כאן אנו מתארים מבחן רחב היקף של ביטוי חלבונים מערכת עובריים אנושיים לתאי כליה 293T, שניתן להשתמש בהם כדי לייצר מגוון של חלבונים רקומביננט?...

Video: A Convenient and General Expression Platform for the Production of Secreted Proteins from Human Cells

The Preparation of Primary Hematopoietic Cell Cultures From Murine Bone Marrow for Electroporation

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser MXcell  electroporation system and Gene Pulser  electroporation buffer were specifically developed to transfect nucleic acids into mammalian cells and difficult-to-transfect cells, such as primary and stem cells.This video demonstrates how to establish primary hematopoietic cell cultures from murine bone marrow, and then prepare them for electroporation in the MXcell system. We begin by isolating femur and tibia. Bone marrow from both femur and tibia are then harvested and cultures are established. Cultured bone marrow cells are then transfected and analyzed.

This procedure describes how to establish primary hematopoietic cell cultures from murine bone marrow and is followed by transfection using the Gene Pulser MXCell electroporation system.

הכנת ראשי בתרביות תאים hematopoietic ממח העצם Murine עבור electroporation

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser ...

Staining of Proteins in Gels with Coomassie G-250 without Organic Solvent and Acetic Acid

In classical protein staining protocols using Coomassie Brilliant Blue (CBB), solutions with high contents of toxic and flammable organic solvents (Methanol, Ethanol or 2-Propanol) and acetic acid are used for fixation, staining and destaining of proteins in a gel after SDS-PAGE. To speed up the procedure, heating the staining solution in the microwave oven for a short time is frequently used. This usually results in evaporation of toxic or hazardous Methanol, Ethanol or 2-Propanol and a strong smell of acetic acid in the lab which should be avoided due to safety considerations. In a protocol originally published in two patent applications by E.M. Wondrak (US2001046709 (A1), US6319720 (B1)), an alternative composition of the staining solution is described in which no organic solvent or acid is used. The CBB is dissolved in bidistilled water (60-80mg of CBB G-250 per liter) and 35 mM HCl is added as the only other compound in the staining solution. The CBB staning of the gel is done after SDS-PAGE and thorough washing of the gel in bidistilled water. By heating the gel during the washing and staining steps, the process can be finished faster and no toxic or hazardous compunds are evaporating. The staining of proteins occurs already within 1 minute after heating the gel in staining solution and is fully developed after 15-30 min with a slightly blue background that is destained completely by prolonged washing of the stained gel in bidistilled water, without affecting the stained protein bands.

A short protocol for protein staining with Coomassie Brilliant Blue (CBB) G-250 in polyacrylamide gels is described without using organic solvents or acetic acid as in the classical staining procedures with CBB.

הכתמה של חלבונים עם ג&#39;ל Coomassie G-250 ללא חומצה אצטית ממס אורגני

In classical protein staining protocols using Coomassie Brilliant Blue (CBB), solutions with high contents of toxic and flammable organic solvents ...

Using the Gene Pulser MXcell Electroporation System to Transfect Primary Cells with High Efficiency

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser MXcell electroporation system and Gene Pulser electroporation buffer (Bio-Rad) were specifically developed to easily transfect nucleic acids into mammalian cells and difficult-to-transfect cells, such as primary and stem cells. We will demonstrate how to perform a simple experiment to quickly identify the best electroporation conditions. We will demonstrate how to run several samples through a range of electroporation conditions so that an experiment can be conducted at the same time as optimization is performed. We will also show how optimal conditions identified using 96-well electroporation plates can be used with standard electroporation cuvettes, facilitating the switch from electroporation plates to electroporation cuvettes while maintaining the same electroporation efficiency. In the video, we will also discuss some of the key factors that can lead to the success or failure of electroporation experiments.

This procedure shows how to use the Gene Pulser MXcell electroporation system to rapidly and easily identify the best electroporation conditions for mouse embryonic fibroblasts (MEFs) or other primary cells. Considerations for troubleshooting are also discussed in the associated video.

שימוש ג&#39;ין Pulser מערכת MXcell electroporation כדי Transfect תאים ראשוניים עם יעילות גבוהה

Using an Automated Cell Counter to Simplify Gene Expression Studies: siRNA Knockdown of IL-4 Dependent Gene Expression in Namalwa Cells

The use of siRNA mediated gene knockdown is continuing to be an important tool in studies of gene expression.  siRNA studies are being conducted not only to study the effects of downregulating single genes, but also to interrogate signaling pathways and other complex interaction networks.  These pathway analyses require both the use of relevant cellular models and methods that cause less perturbation to the cellular physiology.  Electroporation is increasingly being used as an effective way to introduce siRNA and other nucleic acids into difficult to transfect cell lines and primary cells without altering the signaling pathway under investigation. There are multiple critical steps to a successful siRNA experiment, and there are ways to simplify the work while improving the data quality at several experimental stages.  To help you get started with your siRNA mediated gene knockdown project, we will demonstrate how to perform a pathway study complete from collecting and counting the cells prior to electroporation through post transfection real-time PCR gene expression analysis.  The following study investigates the role of the transcriptional activator STAT6 in IL-4 dependent gene expression of CCL17 in a Burkitt lymphoma cell line (Namalwa).  The techniques demonstrated are useful for a wide range of siRNA-based experiments on both adherent and suspension cells.  We will also show how to streamline cell counting with the TC10 automated cell counter, how to electroporate multiple samples simultaneously using the MXcell electroporation system, and how to simultaneously assess RNA quality and quantity with the Experion automated electrophoresis system.

This procedure describes a quick and easy workflow to introduce siRNA into difficult to transfect cell lines and follow gene expression by real-time PCR. Use of an automated cell counter, multi-well electroporation plate, and automated electrophoresis station provide quick and reliable results without the need for expensive robotic handling.

שימוש מונה תא אוטומטי כדי לפשט את הביטוי מחקרים ג&#39;ין: siRNA מציאה הביטוי IL 4-Gene תלויים תאים Namalwa

The use of siRNA mediated gene knockdown is continuing to be an important tool in studies of gene expression.  siRNA studies are being conducted not only ...

The MultiBac Protein Complex Production Platform at the EMBL

Proteomics research revealed the impressive complexity of eukaryotic proteomes in unprecedented detail. It is now a commonly accepted notion that proteins in cells mostly exist not as isolated entities but exert their biological activity in association with many other proteins, in humans ten or more, forming assembly lines in the cell for most if not all vital functions.1,2 Knowledge of the function and architecture of these multiprotein assemblies requires their provision in superior quality and sufficient quantity for detailed analysis. The paucity of many protein complexes in cells, in particular in eukaryotes, prohibits their extraction from native sources, and necessitates recombinant production. The baculovirus expression vector system (BEVS) has proven to be particularly useful for producing eukaryotic proteins, the activity of which often relies on post-translational processing that other commonly used expression systems often cannot support.3 BEVS use a recombinant baculovirus into which the gene of interest was inserted to infect insect cell cultures which in turn produce the protein of choice. MultiBac is a BEVS that has been particularly tailored for the production of eukaryotic protein complexes that contain many subunits.4 A vital prerequisite for efficient production of proteins and their complexes are robust protocols for all steps involved in an expression experiment that ideally can be implemented as standard operating procedures (SOPs) and followed also by non-specialist users with comparative ease. The MultiBac platform at the European Molecular Biology Laboratory (EMBL) uses SOPs for all steps involved in a multiprotein complex expression experiment, starting from insertion of the genes into an engineered baculoviral genome optimized for heterologous protein production properties to small-scale analysis of the protein specimens produced.5-8 The platform is installed in an open-access mode at EMBL Grenoble and has supported many scientists from academia and industry to accelerate protein complex research projects.

Protein complexes catalyze key cellular functions. Detailed functional and structural characterization of many essential complexes requires recombinant production. MultiBac is a baculovirus/insect cell system particularly tailored for expressing eukaryotic proteins and their complexes. MultiBac was implemented as an open-access platform, and standard operating procedures developed to maximize its utility.

פלטפורמת הפקת MultiBac החלבון המורכבת בEMBL

Proteomics  research revealed the impressive complexity of eukaryotic proteomes in  unprecedented detail. It is now a commonly accepted notion that ...

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 ...

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 ...

Enrichment of Extracellular Matrix Proteins from Tissues and Digestion into Peptides for Mass Spectrometry Analysis

The extracellular matrix (ECM) is a complex meshwork of cross-linked proteins that provides biophysical and biochemical cues that are major regulators of cell proliferation, survival, migration, etc. The ECM plays important roles in development and in diverse pathologies including cardio-vascular and musculo-skeletal diseases, fibrosis, and cancer. Thus, characterizing the composition of ECMs of normal and diseased tissues could lead to the identification of novel prognostic and diagnostic biomarkers and potential novel therapeutic targets. However, the very nature of ECM proteins (large in size, cross-linked and covalently bound, heavily glycosylated) has rendered biochemical analyses of ECMs challenging. To overcome this challenge, we developed a method to enrich ECMs from fresh or frozen tissues and tumors that takes advantage of the insolubility of ECM proteins. We describe here in detail the decellularization procedure that consists of sequential incubations in buffers of different pH and salt and detergent concentrations and that results in 1) the extraction of intracellular (cytosolic, nuclear, membrane and cytoskeletal) proteins and 2) the enrichment of ECM proteins. We then describe how to deglycosylate and digest ECM-enriched protein preparations into peptides for subsequent analysis by mass spectrometry.

This protocol describes a procedure for enriching ECM proteins from tissues or tumors and deglycosylating and digesting the ECM-enriched preparations into peptides to analyze their protein composition by mass spectrometry.

העשרה תאית מטריקס חלבונים מרקמות ועיכול לפפטידים לניתוח ספקטרומטריית המסה

The extracellular matrix (ECM) is a complex meshwork of cross-linked proteins that provides biophysical and biochemical cues that are major regulators of ...

High Yield Expression of Recombinant Human Proteins with the Transient Transfection of HEK293 Cells in Suspension

The art of producing recombinant proteins with complex post-translational modifications represents a major challenge for studies of structure and function. The rapid establishment and high recovery from transiently-transfected mammalian cell lines addresses this barrier and is an effective means of expressing proteins that are naturally channeled through the ER and Golgi-mediated secretory pathway. Here is one protocol for protein expression using the human HEK293F and HEK293S cell lines transfected with a mammalian expression vector designed for high protein yields. The applicability of this system is demonstrated using three representative glycoproteins that expressed with yields between 95-120 mg of purified protein recovered per liter of culture. These proteins are the human Fc&#947;RIIIa and the rat &#945;2-6 sialyltransferase, ST6GalI, both expressed with an N-terminal GFP fusion, as well as the unmodified human immunoglobulin G1 Fc. This robust system utilizes a serum-free medium that is adaptable for expression of isotopically enriched proteins and carbohydrates for structural studies using mass spectrometry and nuclear magnetic resonance spectroscopy. Furthermore, the composition of the N-glycan can be tuned by adding a small molecule to prevent certain glycan modifications in a manner that does not reduce yield.

Laboratory-scale production of eukaryotic proteins with appropriate post-translational modification represents a significant barrier. Here is a robust protocol with rapid establishment and turnaround for protein expression using a mammalian expression system. This system supports selective amino acid, selective labeling of proteins and small molecule modulators of glycan composition.

ביטוי תשואה גבוהה של חלבונים אנושיים רקומביננטי עם transfection חולף של תאי HEK293 בהשעיה

The art of producing recombinant proteins with complex post-translational modifications represents a major challenge for studies of structure and ...

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 ...