עבודה זו נתמכה על ידי פרס CA116429 K08-04 ל MC ועל ידי מענק מטעם סרטן לכבוש את עכשיו (הנוגעים) תשתית # 8501

<ol>
	<li>Camps, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modulation+of+ColE1-like+plasmid+replication+for+recombinant+gene+expression.">Modulation of ColE1-like plasmid replication for recombinant gene expression.</a> Recent Pat DNA Gene Seq. 4, 58-73 (2010).</li><li>Itoh, T., Tomizawa, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Initiation+of+replication+of+plasmid+ColE1+DNA+by+RNA+polymerase,+ribonuclease+H,+and+DNA+polymerase+I.">Initiation of replication of plasmid ColE1 DNA by RNA polymerase, ribonuclease H, and DNA polymerase I.</a> Cold Spring Harb Symp Quant Biol. 43 Pt 1, 409-417 (1979).</li><li>Polisky, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ColE1+replication+control+circuitry:+sense+from+antisense.">ColE1 replication control circuitry: sense from antisense.</a> Cell. 55, 929-932 (1988).</li><li>Camps, M., Naukkarinen, J., Johnson, B. P., Loeb, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeted+gene+evolution+in+Escherichia+coli+using+a+highly+error-prone+DNA+polymerase+I.">Targeted gene evolution in Escherichia coli using a highly error-prone DNA polymerase I.</a> Proc Natl Acad Sci U S A. 100, 9727-9732 (2003).</li><li>Shinkai, A., Loeb, L. 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+mutagenesis+by+Escherichia+coli+DNA+polymerase+I.+Ile(709)+in+motif+A+functions+in+base+selection.">In vivo mutagenesis by Escherichia coli DNA polymerase I. Ile(709) in motif A functions in base selection.</a> J Biol Chem. 276, 46759-46764 (2001).</li><li>Uyemura, D., Lehman, I. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biochemical+characterization+of+mutant+forms+of+DNA+polymerase+I+from+Escherichia+coli.+I.+The+polA12+mutation.">Biochemical characterization of mutant forms of DNA polymerase I from Escherichia coli. I. The polA12 mutation.</a> J Biol Chem. 251, 4078-4084 (1976).</li><li>Sedgwick, B., Robins, P., Lindahl, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+removal+of+alkylation+damage+from+DNA+by+AlkB+and+related+DNA+dioxygenases.">Direct removal of alkylation damage from DNA by AlkB and related DNA dioxygenases.</a> Methods Enzymol. 408, 108-120 (2006).</li><li>Aharoni, A., Gaidukov, L., Khersonsky, O., Mc, Q. G. S., Roodveldt, C., Tawfik, D. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+'evolvability'+of+promiscuous+protein+functions.">The 'evolvability' of promiscuous protein functions.</a> Nat Genet. 37, 73-76 (2005).</li><li>Elena, S. F., Lenski, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evolution+experiments+with+microorganisms:+the+dynamics+and+genetic+bases+of+adaptation.">Evolution experiments with microorganisms: the dynamics and genetic bases of adaptation.</a> Nat Rev Genet. 4, 457-469 (2003).</li><li>Itoh, T., Tomizawa, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=FoFormation+of+an+RNA+primer+for+initiation+of+replication+of+ColE1+DNA+by+ribonuclease+H.">FoFormation of an RNA primer for initiation of replication of ColE1 DNA by ribonuclease H.</a> Proc Natl Acad Sci U S A. 77, 2450-2454 (1980).</li><li>Bryan, S. K., Moses, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sufficiency+of+the+Klenow+fragment+for+survival+of+polC(Ts)+pcbA1+Escherichia+coli+at+43+degrees.">Sufficiency of the Klenow fragment for survival of polC(Ts) pcbA1 Escherichia coli at 43 degrees.</a> C. J Bacteriol. 170, 456-458 (1988).</li></ol>

אין ניגודי אינטרסים הכריז.

מאמר זה מציג פרוטוקול mutagenesis המאפשר את הדור של ספריות גדולות מוטציה אקראית ללא צורך בשיבוט או PCR. שיטה זו מבוססת על שכפול וטעייה מועדים של קידוד פלסמיד רצף של עניין. בתיאוריה, מוטציות יש להגביל במידה רבה 100-300 נ&quot;ב ממוקם מיד במורד הזרם של הבורר RNA / DNA, בגודל של מנהיג גדיל ביניים הופק על ידי פול אני 2,10. מצאנו כי בתנאים שהוצגו פרוטוקול זה, פול מוטציות אני להתרחש בכל רחבי הפלסמיד, אם כי בתדירות הולכת ופוחתת ככל המרחק גדל פלסמיד אורי (איור 3). ממצא זה מרמז כי המעבר או &quot;הבורר&quot; של פול אני אל Pol III במהלך שכפול ColE1 פלסמיד הוא הרבה יותר הדרגתית מאשר שדווח קודם לכן, לפחות בתנאי הניסוי שלנו 2, ומסכים עם מחקרים קודמים מציע יתירות פונקציונלית בין Pol Pol ו אני III 11. הפרוטוקול המקורי שלנו תיאר mutagenesis בתרבויות נוזלי (4). פרוטוקול זה מניב 0.41 מוטציות / kb עבור d &lt;1000, כלומר בתוך 1000 bp סמוך RNA / DNA מתג (טבלה 1). Hypersaturation ידי עזיבת תרבות נוזל בתוך שייקר במשך 3 ימים ללא תוספת של כל מדיה טרי מעלה את שכיחות המוטציה על 0.70 מוטציות / kb אבל התוצאה היא תשואה פלסמיד עני מאוד (פחות מ -1% לעומת יום 1; מידע לא מוצג) ( טבלה 1). כאן אנו מציגים פרוטוקול פשוט המבוסס על ציפוי ישירות את הטרנספורמציה של יעד פלסמיד על אגר מוצק ב 37 ° C (איור 1). פרוצדורה זו מייצרת את תדר הגדול ביותר של מוטציה / מעגל mutagenesis (0.92 מוטציות / kb עבור d &lt;1000) ו מאוד מקל איטרציה. שינוי התנאים תרבות התקשורת מוצק משפיע גם על ספקטרום מוטציה (טבלה 2). Mutagenesis ציפוי ישיר מפחית סימנה את האסימטריה בין משלים צמד החלפות בסיס לראות תרבות נוזלי (להשוות C → T לעומת G → ​​A ו-G מול → T → C). מצד שני, ציפוי ישיר מיוצר indels יותר (0.5% פחות מאשר 4%) וירידה במספר מוטציות G → ​​ידי 3-לקפל, המובילה לייצוג יתר של G / C מוטציות (74%). בסך הכל, אנו מאמינים כי פשטות ויעילות מוגברת של פרוטוקול ציפוי ישיר שהוצגו כאן עולה על היתרונות של ספקטרום מוטציה מעט מאוזן יותר המיוצר על ידי mutagenesis נוזלי. במגבלת היעד של פלסמיד, מוטציות הנוצר על ידי השיטה שלנו לא מוגבלים רצף היעד הרצוי. עם זאת, ההתפתחות של פעילויות ביוכימיות רומן צריך להיות תלוי מוטציות בתוך הגן היעד, שכן היא מייצגת איכותי ולא שינוי כמותי. לפיכך, שילוב mutagenesis הפלסמיד שלנו עם הקרנה של מוטציות על צלחות שיפוע מנצל את היתרונות של המערכת שלנו (ספריות גדולות וזמינות של מבחר) לאבולוציה של מאפיינים ביוכימיים חדשים. יישומים אחרים של mutagenesis אקראי כגון אופטימיזציה של הפעילות האנזימטית קיימים או אקראי אזורים ספציפיים של גן דורשים שיבוט הבאים mutagenesis אקראי. במקרה זה, בעל ספריה הפלסמיד לעומת מוצר הגברה PCR משפר את יעילות שיבוט ידי הקלת הגברה הגבלה. לסיכום, אנחנו מדגימים את פרוטוקול פשוט ליצור ספריה מוטציה אקראית עבור גן מטרה בהתחשב בעובדה בולטת בפשטותה לגיוון של ספריות שנוצר. אנו מראים כיצד שיטה זו יכולה להיות יחד עם בחירות פונקציונלי לאבולוציה יעיל של פעילויות ביוכימיות חדש. בנוסף, שלנו vivo שנוצר ספריות ניתן לשכפל בקלות, ומאפשר לאתר ספציפי mutagenesis או אופטימיזציה של הפעילות הקיימת.

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		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>carbenicillin</td>
<td>&nbsp;</td>
<td>CellGro</td>
<td>46100R6</td>
<td>0.1mg/ml final</td>
</tr>
<tr>
<td>tetracycline</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>BP7640</td>
<td>0.05mg/ml final</td>
</tr>
<tr>
<td>chloramphenicol</td>
<td>&nbsp;</td>
<td>Genlantis</td>
<td>M120100</td>
<td>0.03mg/ml final</td>
</tr>
<tr>
<td>LB Agar Miller</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>BP1425</td>
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<td>LB Broth Miller</td>
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<td>Difco</td>
<td>244620</td>
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<td>Soft Agar</td>
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<td>Difco</td>
<td>214580</td>
<td>Made in house</td>
</tr>
<tr>
<td>Acridine Orange</td>
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<td>Sigma</td>
<td>A38401-1</td>
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<td>Antifoam B emulsion</td>
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<td>Sigma</td>
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<tr>
<td>Glycerol</td>
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<td>332030025</td>
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<td>100x100x15mm sq dish</td>
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<td>Fisher</td>
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<td>100mm rnd dish </td>
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<td>Fisher</td>
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<td>&nbsp;</td>
<tr>
<td>Microscope slide 25x75x1mm</td>
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<td>Gold Seal</td>
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<td>Electroporator 2510</td>
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<td>Electroporator 2510</td>
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<td>Eppendorf</td>
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<td>2mm gap tubes</td>
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<td>MolBioproducts</td>
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<td>Zippy mini prep kit</td>
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<td>Zymo </td>
<td>D4020</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

mutagenesis and functional selection protocols for directed evolution of proteins in e. coli

הדור יעיל של המגוון הגנטי מייצג כלי מולקולרית יסולא בפז כי ניתן להשתמש בתווית סינתזה של DNA, כדי ליצור חתימות מולקולרי ייחודי, או להתפתח חלבונים במעבדה. כאן, אנו מציגים פרוטוקול המאפשר דור (&gt; 10 11) ספריות גדולות מוטציה עבור רצף יעד נתון. שיטה זו מבוססת על שכפול של קידוד ColE1 פלסמיד רצף הרצוי על ידי גרסה נמוכה נאמנות של פולימראז ה-DNA אני (LF-Pol אני). היעד פלסמיד הופכת זן המוטטורי של E. coli ו מצופה על התקשורת מוצק, מניב בין 0.2 ל 1 מוטציות / kb, בהתאם למיקום של גן המטרה. תדרים מוטציה גבוהה מושגות על ידי iterating תהליך זה של mutagenesis. בהשוואה לשיטות חלופיות mutagenesis, פרוטוקול שלנו בולטת בפשטותה, שכן לא שיבוט PCR או מעורבים. לכן, השיטה שלנו הוא אידיאלי עבור תיוג מוטאציות של פלסמידים או אחרת אני תבניות פול או לחקור חלקים גדולים של מרחב רצף לאבולוציה של פעילויות לא נוכח היעד המקורי. השליטה במרחב הדוק כי PCR או להציע אקראי oligonucleotide שיטות המבוססות יכול גם להיות מושגת באמצעות שיבוט הבאות של חלקים מסוימים של הספרייה. כאן אנו מספקים פרוטוקולים מראה כיצד ליצור ספריה מוטציה אקראית וכיצד להקים התרופה מבוססת בחירות E. coli כדי לזהות מוטציות מציג פעילויות ביוכימיות חדש.

Watch this Scientific Journal Video about Mutagenesis and Functional Selection Protocols for Directed Evolution of Proteins in E. coli at JoVE.com

Mutagenesis and Functional Selection Protocols for Directed Evolution of Proteins in E. coli

כאן אנו להדגים פרוטוקול פשוט ליצור ספריה מוטציה אקראית על רצף יעד נתון. אנו מראים כיצד שיטה זו, אשר מבוצעת in vivo של Escherichia coli, יכול להיות יחד עם בחירות פונקציונלי להתפתח פעילות אנזימטית חדשה.

בחירת הפרוטוקולים mutagenesis ו פונקציונאלי אבולוציה מכוונת של חלבונים א coli</em

The efficient generation of genetic diversity represents an invaluable molecular tool that can be used to label DNA synthesis, to create unique molecular ...

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Mutagenesis and Functional Selection Protocols for Directed Evolution of Proteins in E. coli

30.2K Views.  University of California Santa Cruz - UCSC. כאן אנו להדגים פרוטוקול פשוט ליצור ספריה מוטציה אקראית על רצף יעד נתון. אנו מראים כיצד שיטה זו, אשר מבוצעת in vivo של Escherichia coli, יכול להיות יחד עם בחירות פונקציונלי להתפתח פעילות אנזימטית חדשה.

Video: Mutagenesis and Functional Selection Protocols for Directed Evolution of Proteins in E. coli

Transformation of Plasmid DNA into E. coli Using the Heat Shock Method

Transformation of plasmid DNA into E. coli using the heat shock method is a basic technique of molecular biology. It consists of inserting a foreign plasmid or ligation product into bacteria. This video protocol describes the traditional method of transformation using commercially available chemically competent bacteria from Genlantis. After a short incubation in ice, a mixture of chemically competent bacteria and DNA is placed at 42&deg;C for 45 seconds (heat shock) and then placed back in ice. SOC media is added and the transformed cells are incubated at 37&deg;C for 30 min with agitation. To be assured of isolating colonies irrespective of transformation efficiency, two quantities of transformed bacteria are plated. This traditional protocol can be used successfully to transform most commercially available competent bacteria. The turbocells from Genlantis can also be used in a novel 3-minute transformation protocol, described in the instruction manual.

טרנספורמציה של DNA פלסמיד לתוך החיידק שימוש בשיטה הלם חום

Transformation of plasmid DNA into E. coli using the heat shock method is a basic technique of molecular biology. It consists of inserting a foreign ...

Homemade Site Directed Mutagenesis of Whole Plasmids

Site directed mutagenesis of whole plasmids is a simple way to create slightly different variations of an original plasmid. With this method the cloned target gene can be altered by substitution, deletion or insertion of a few bases directly into a plasmid. It works by simply amplifying the whole plasmid, in a non PCR-based thermocycling reaction. During the reaction mutagenic primers, carrying the desired mutation, are integrated into the newly synthesized plasmid. In this video tutorial we demonstrate an easy and cost effective way to introduce base substitutions into a plasmid. The protocol works with standard reagents and is independent from commercial kits, which often are very expensive. Applying this protocol can reduce the total cost of a reaction to an eighth of what it costs using some of the commercial kits. In this video we also comment on critical steps during the process and give detailed instructions on how to design the mutagenic primers.

Site directed mutagenesis of whole plasmids is a simple way to create slightly different variations of an original plasmid. Here we demonstrate an easy and cost effective way to introduce base substitutions into a plasmid using standard reagents.

Mutagenesis האתר ביתי בימוי של פלסמידים שלמים

Site directed mutagenesis of whole plasmids is a simple way to create slightly different variations of an original plasmid. With this method the cloned ...

Crystallizing Membrane Proteins for Structure Determination using Lipidic Mesophases

A detailed protocol for crystallizing membrane proteins by using lipidic mesophases is described. This method has variously been referred to as the lipidic cubic phase or in meso method. The method has been shown to be quite versatile in that it has been used to solve X-ray crystallographic structures of prokaryotic and eukaryotic proteins, proteins that are monomeric, homo- and hetero-multimeric, chromophore-containing and chromophore-free, and alpha-helical and beta-barrel proteins. Recent successes using in meso crystallization are the human engineered beta2-adrenergic and adenosine A2a G protein-coupled receptors. Protocols are presented for reconstituting the membrane protein into the monoolein-based mesophase, and for setting up crystallizations in the manual mode. Additional steps in the overall process, such as crystal harvesting, are to be addressed in future video articles. The time required to prepare the protein-loaded mesophase and to set up a crystallization plate manually is about one hour.

Herein is described the procedure implemented in the Caffrey Membrane Structural and Functional Biology Group to set up manually crystallization trials of membrane proteins in lipidic mesophases.

גיבוש חלבונים בממברנה עבור קביעת מבנה באמצעות Mesophases Lipidic

A detailed protocol for crystallizing membrane proteins by using lipidic mesophases is described. This method has variously been referred to as the ...

Mutagenesis and Functional Analysis of Ion Channels Heterologously Expressed in Mammalian Cells

We will demonstrate how to study the functional effects of introducing a point mutation in an ion channel. We study G protein-gated inwardly rectifying potassium (referred to as GIRK) channels, which are important for regulating the excitability of neurons. There are four different mammalian GIRK channel subunits (GIRK1-GIRK4) - we focus on GIRK2 because it forms a homotetramer. Stimulation of different types of G protein-coupled receptors (GPCRs), such as the muscarinic receptor (M2R), leads to activation of GIRK channels. Alcohol also directly activates GIRK channels. We will show how to mutate one amino acid by specifically changing one or more nucleotides in the cDNA for the GIRK channel. This mutated cDNA sequence will be amplified in bacteria, purified, and the presence of the point mutation will be confirmed by DNA sequencing. The cDNAs for the mutated and wild-type GIRK channels will be transfected into human embryonic kidney HEK293T cells cultured in vitro. Lastly, whole-cell patch-clamp electrophysiology will be used to study the macroscopic potassium currents through the ectopically expressed wild-type or mutated GIRK channels. In this experiment, we will examine the effect of a L257W mutation in GIRK2 channels on M2R-dependent and alcohol-dependent activation.

We will demonstrate how to study the effect of a single point mutation on the function of an ion channel.

Mutagenesis וניתוח פונקציונלי של תעלות יונים הביעו Heterologously בתאי יונקים

We will demonstrate how to study the functional effects of introducing a point mutation in an ion channel. We study G protein-gated inwardly rectifying ...

Examining the Conformational Dynamics of Membrane Proteins in situ with Site-directed Fluorescence Labeling

Two electrode voltage clamp electrophysiology (TEVC) is a powerful tool to investigate the mechanism of ion transport1 for a wide variety of membrane proteins including ion channels2, ion pumps3, and transporters4. Recent developments have combined site-specific fluorophore labeling alongside TEVC to 
concurrently examine the conformational dynamics at specific residues and function of these proteins on the surface of single cells.
We will describe a method to study the conformational dynamics of membrane proteins by simultaneously monitoring fluorescence and current changes using voltage-clamp fluorometry. This approach can be used to examine the molecular motion of membrane proteins site-specifically following cysteine replacement and site-directed fluorophore labeling5,6. Furthermore, this method provides an approach to determine distance constraints between specific residues7,8. 
This is achieved by selectively attaching donor and acceptor fluorophores to two mutated cysteine residues of interest.
In brief, these experiments are performed following functional expression of the desired protein on the surface of Xenopus leavis oocytes. The large surface area of these oocytes enables facile functional measurements and a robust fluorescence signal5. It is also possible to readily change the extracellular conditions such as pH, ligand or cations/anions, which can provide further information on the mechanism of membrane proteins4. Finally, recent developments 
have also enabled the manipulation of select internal ions following co-expression with a second protein9.
Our protocol is described in multiple parts. First, cysteine scanning mutagenesis proceeded by fluorophore labeling is completed at residues located at the interface of the transmembrane and extracellular domains. Subsequent experiments are designed to identify residues which demonstrate large changes in fluorescence intensity (&lt;5%)3 upon a conformational change of the protein. Second, these changes in fluorescence intensity are compared to the kinetic parameters of 
the membrane protein in order to correlate the conformational dynamics to the function of the protein10. This enables a rigorous biophysical analysis of the molecular motion of the target protein. Lastly, two residues of the holoenzyme can be labeled with a donor and acceptor fluorophore in order to determine distance constraints using donor photodestruction methods. It is also possible to monitor the relative movement of protein subunits following labeling with a donor and acceptor fluorophore.

Examining the Conformational Dynamics of Membrane Proteins in situ with Site-directed Fluorescence Labeling

We will describe a method which measures the kinetics of ion transport of membrane proteins alongside site-specific analysis of conformational changes using fluorescence on single cells. This technique is adaptable to ion channels, transporters and ion pumps and can be utilized to determine distance constraints between protein subunits.

בחינת Dynamics קונפורמציה של חלבונים בממברנה באתרו עם תיוג, האתר מכוון פלואורסצנטי

Two electrode voltage clamp electrophysiology (TEVC) is a powerful tool to investigate the mechanism of ion transport1 for a wide variety of membrane ...

Use of a Robot for High-throughput Crystallization of Membrane Proteins in Lipidic Mesophases

Structure-function studies of membrane proteins greatly benefit from having available high-resolution 3-D structures of the type provided through macromolecular X-ray crystallography (MX). An essential ingredient of MX is a steady supply of ideally diffraction-quality crystals. The in meso or lipidic cubic phase (LCP) method for crystallizing membrane proteins is one of several methods available for crystallizing membrane proteins. It makes use of a bicontinuous mesophase in which to grow crystals. As a method, it has had some spectacular successes of late and has attracted much attention with many research groups now interested in using it. One of the challenges associated with the method is that the hosting mesophase is extremely viscous and sticky, reminiscent of a thick toothpaste. Thus, dispensing it manually in a reproducible manner in small volumes into crystallization wells requires skill, patience and a steady hand. A protocol for doing just that was developed in the Membrane Structural &amp; Functional Biology (MS&amp;FB) Group1-3. JoVE video articles describing the method are available1,4. 
The manual approach for setting up in meso trials has distinct advantages with specialty applications, such as crystal optimization and derivatization. It does however suffer from being a low throughput method. Here, we demonstrate a protocol for performing in meso crystallization trials robotically. A robot offers the advantages of speed, accuracy, precision, miniaturization and being able to work continuously for extended periods under what could be regarded as hostile conditions such as in the dark, in a reducing atmosphere or at low or high temperatures. An in meso robot, when used properly, can greatly improve the productivity of membrane protein structure and function research by facilitating crystallization which is one of the slow steps in the overall structure determination pipeline. 
In this video article, we demonstrate the use of three commercially available robots that can dispense the viscous and sticky mesophase integral to in meso crystallogenesis. The first robot was developed in the MS&amp;FB Group5,6. The other two have recently become available and are included here for completeness.	
An overview of the protocol covered in this article is presented in Figure 1. All manipulations were performed at room temperature (~20 &deg;C) under ambient conditions.

Herein is described a robotic approach to high-throughput crystallization of membrane proteins in lipidic mesophases for use in structure determination using macromolecular X-ray crystallography. Three robots capable of handling the viscous and sticky protein-laden mesophase integral to the method are introduced.

שימוש ברובוט להתגבשות תפוקה גבוהה של חלבונים בממברנה בMesophases Lipidic

Structure-function studies of membrane proteins greatly benefit from having available high-resolution 3-D structures of the type provided through ...

Protocols for Implementing an Escherichia coli Based TX-TL Cell-Free Expression System for Synthetic Biology

Ideal cell-free expression systems can theoretically emulate an in vivo cellular environment in a controlled in vitro platform.1 This is useful for expressing proteins and genetic circuits in a controlled manner as well as for providing a prototyping environment for synthetic biology.2,3 To achieve the latter goal, cell-free expression systems that preserve endogenous Escherichia coli transcription-translation mechanisms are able to more accurately reflect in vivo cellular dynamics than those based on T7 RNA polymerase transcription. We describe the preparation and execution of an efficient endogenous E. coli based transcription-translation (TX-TL) cell-free expression system that can produce equivalent amounts of protein as T7-based systems at a 98% cost reduction to similar commercial systems.4,5 The preparation of buffers and crude cell extract are described, as well as the execution of a three tube TX-TL reaction. The entire protocol takes five days to prepare and yields enough material for up to 3000 single reactions in one preparation. Once prepared, each reaction takes under 8 hr from setup to data collection and analysis. Mechanisms of regulation and transcription exogenous to E. coli, such as lac/tet repressors and T7 RNA polymerase, can be supplemented.6 Endogenous properties, such as mRNA and DNA degradation rates, can also be adjusted.7 The TX-TL cell-free expression system has been demonstrated for large-scale circuit assembly, exploring biological phenomena, and expression of proteins under both T7- and endogenous promoters.6,8 Accompanying mathematical models are available.9,10 The resulting system has unique applications in synthetic biology as a prototyping environment, or "TX-TL biomolecular breadboard."

Protocols for Implementing an Escherichia coli Based TX-TL Cell-Free Expression System for Synthetic Biology

This five-day protocol outlines all steps, equipment, and supplemental software necessary for creating and running an efficient endogenous Escherichia coli based TX-TL cell-free expression system from scratch. With reagents, the protocol takes 8 hours or less to setup a reaction, collect, and process data.

פרוטוקולים ליישום<em&gt; חיידקי Escherichia</emמערכת&gt; בהתבסס TX-TL סלולרי ללא ביטוי של ביולוגיה סינתטית

Ideal cell-free expression systems can theoretically emulate an in vivo cellular environment in a controlled in vitro platform.1 This is useful for ...

High Throughput Quantitative Expression Screening and Purification Applied to Recombinant Disulfide-rich Venom Proteins Produced in E. coli

Escherichia coli (E. coli) is the most widely used expression system for the production of recombinant proteins for structural and functional studies. However, purifying proteins is sometimes challenging since many proteins are expressed in an insoluble form. When working with difficult or multiple targets it is therefore recommended to use high throughput (HTP) protein expression screening on a small scale (1-4&#160;ml cultures) to quickly identify conditions for soluble expression. To cope with the various structural genomics programs of the lab, a quantitative (within a range of 0.1-100 mg/L culture of recombinant protein) and HTP protein expression screening protocol was implemented and validated on thousands of proteins. The protocols were automated with the use of a liquid handling robot but can also be performed manually without specialized equipment.
Disulfide-rich venom proteins are gaining increasing recognition for their potential as therapeutic drug leads. They can be highly potent and selective, but their complex disulfide bond networks make them challenging to produce. As a member of the FP7 European Venomics project (<a href="http://www.venomics.eu">www.venomics.eu</a>), our challenge is to develop successful production strategies with the aim of producing thousands of novel venom proteins for functional characterization. Aided by the redox properties of disulfide bond isomerase DsbC, we adapted our HTP production pipeline for the expression of oxidized, functional venom peptides in the E. coli cytoplasm. The protocols are also applicable to the production of diverse disulfide-rich proteins. Here we demonstrate our pipeline applied to the production of animal venom proteins. With the protocols described herein it is likely that soluble disulfide-rich proteins will be obtained in as little as a week. Even from a small scale, there is the potential to use the purified proteins for validating the oxidation state by mass spectrometry, for characterization in pilot studies, or for sensitive micro-assays.

High Throughput Quantitative Expression Screening and Purification Applied to Recombinant Disulfide-rich Venom Proteins Produced in E. coli

A protocol for the quantitative, high throughput expression screening and analytical purification of fusion proteins from small-scale Escherichia coli cultures is described and applied to the expression of disulfide-rich animal venom protein targets.

הקרנת תפוקה גבוהה ביטוי כמותי וטיהור יישומים לחלבוני ארס רקומביננטי דיסולפיד עשירים הופק ב<em&gt; א coli</em

Escherichia coli (E. coli) is the most widely used expression system for the production of recombinant proteins for structural and functional studies. ...

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 ...

Functional Reconstitution and Channel Activity Measurements of Purified Wildtype and Mutant CFTR Protein

The Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) is a unique channel-forming member of the ATP Binding Cassette (ABC) superfamily of transporters. The phosphorylation and nucleotide dependent chloride channel activity of CFTR has been frequently studied in whole cell systems and as single channels in excised membrane patches. Many Cystic Fibrosis-causing mutations have been shown to alter this activity. While a small number of purification protocols have been published, a fast reconstitution method that retains channel activity and a suitable method for studying population channel activity in a purified system have been lacking. Here rapid methods are described for purification and functional reconstitution of the full-length CFTR protein into proteoliposomes of defined lipid composition that retains activity as a regulated halide channel. This reconstitution method together with a novel flux-based assay of channel activity is a suitable system for studying the population channel properties of wild type CFTR and the disease-causing mutants F508del- and G551D-CFTR. Specifically, the method has utility in studying the direct effects of phosphorylation, nucleotides and small molecules such as potentiators and inhibitors on CFTR channel activity. The methods are also amenable to the study of other membrane channels/transporters for anionic substrates.

Described here is a rapid and effective procedure for functional reconstitution of purified wild-type and mutant CFTR protein that preserves activity for this chloride channel, which is defective in Cystic Fibrosis. Iodide efflux from reconstituted proteoliposomes mediated by CFTR allows studies of channel activity and the effects of small molecules.