עבודה זו נתמכה בחלקה על ידי מענק קרנות לתשתיות למחקר (RSFG) מאוניברסיטת אינדיאנה-Purdue University, אינדיאנפוליס, ובחלקה על ידי בפרס של איגוד הלב האמריקאי 14GRNT20390154, כדי ARK

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The authors have nothing to disclose.

אנו מדגימים את היתרון של גישה משולבת לניתוח הרכבה הפרוטאזום ידי nondenaturing עמוד באמצעות פרוטאזומים archaeal רקומביננטי. 9,11 השיטה הרגילה של Coexpression בקטריאלי של יחידות משנה הפרוטאזום מאפשר ניתוח מהיר אבל לא יכול לחשוף ביניים הרכבה מפתח. אנו מציעים שילוב Coexpression עם lysate ער...

מתחמי multiprotein לבצע פעילות הסלולר קריטית רבה 1. עבור רבי קומפלקסים אלה, הרבה יותר ידוע על המבנה שלהם ותפקוד מאשר על ההרכבה שלהם 2,3. הפרוטאזום הוא מורכב אחד כזה והוא נמצא בכל תחומי החיים. אאוקריוטים, מכונה מולקולרית זה הוא הליבה של המערכת הפרוטסומית / יוביקוויטין (UPS) ומספק את המסלול העיקרי של פירוק חלבונים תאיים 4. הפרוטאזום איקריוטיים (המכונה הפרוטאזום 26s) מורכב משני מכלולים תת מרכזיים: א הפרוטאזום 20S, או חלקיקי Core (CP) 5, שניתן כתרים על קצוות אחד או שניהם על ידי חלקיק 19S תקין (RP) 6. הפרוטאזום 20S הוא פרוטאז מידור גדול. המבנה הרביעון שלה נשמר לחלוטין בכל תחומי חיים והוא מורכב ערימה של ארבע טבעות שבע membered המכיל שני סוגים של יחידות משנה הקשורות מבני, α; ובטא 5,7,8. אאוקריוטים, שתי הטבעות החיצוניות כל מונה שבע יחידות משנת α ברורות ואת שתי הטבעות פנימיות כל מונה שבע יחידות משנת β ברורות; פעילות פרוטאוליטים מתגוררת בתוך שלוש של יחידות מהשנה β. לעומת זאת, טבעות CP של קדומים וחיידקים הן בדרך כלל מורכבות רק סוג אחד של α וסוג אחד של למקטע β. פרוטאזומים Archaeal סיפקו מערכת מודל חשוב ללמוד הרכבה הפרוטאזום הן בשל פשטות ההלחנה שלהם ושיתוף שלהם מנגנון הרכבה משותף עם עמיתיהם איקריוטיים שלהם 9-13. בקיצור, יחידות משנה α להרכיב לטבעות α ראשונה, אשר לשמש פיגום שעליו בטא יחידות משנה להרכיב. וכתוצאה מכך חצי פרוטאזומים (α 7 β 7) dimerize, והוליד התאספו מלא CP (α 7 β 7 β 7 α 7). במהלך dimerization, את propeptides להציג על יחידות משנה בטאיוסרו autocatalytically, חשיפת threonines N-terminal קטליטי. השימוש פרוטאזומים archaeal מודל הרכבה לעתים קרובות מנצל את הייצור של חלבונים הפרוטאזום archaeal רקומביננטי coli Escherichia. זוהי גישה כדאית משום שהוא מאפשר יחידות משנה להיות מיוצר בצירופים שונים, כמו גם WT וגרסאות מוטציה, בתוך הפונדקאי כי אינו מייצר פרוטאזומים משלה. ניטור ההרכבה של קומפלקסי חלבונים רבים ביוכימית דורש סוג כלשהו של שיטת חלוקת המפריד מתחמים מורכבים במלואו מן ביניים ומבשרי הרכבה. בשל יכולת בפתרון מעולה, nondenaturing polyacrylamide ג&#39;ל אלקטרופורזה (עמוד) הוכיח להיות שימושי במיוחד החלוקה של מתחמי multiprotein גדולים השונים 14-17. השילוב של ייצור הפרוטאזום archaeal רקומביננטי עמוד nondenaturing הפכה גישה חזקה ב dissectinהפרוטאזום גרם הרכבת 9,11,12,18. עם זאת, השיטה הרגילה על ידי איזו גישה זו מיושמת (כלומר. דרך Coexpression רקומביננטי של יחידות משנת α ו β) יש חסרון חשוב. תגובות עצרת מאורגנות על בסיס שיתופיות וריכוז-תלוי מאוד 3. בהתחשב בכך את ריכוז החלבון בתוך התאים הוא גבוה מאוד 19, בשל תופעות נפח נשלל, תגובות הרכבה להמשיך במהירות in vivo. מכאן אפשר לפספס ביניים הרכבה חשובים כאשר יחידות משנת α ו β הן coexpressed. כאן, אנו טוענים בגישה המשולבת במחקר של הרכבה הפרוטאזום באמצעות יחידות משנה הפרוטאזום archaeal רקומביננטי. לפי גישה זו, הן שיטות ערבוב Coexpression ו lysate מועסקים. הראשון מאפשר ניתוח מהיר של הרכבה כי Coexpression הוא פחות עבודה אינטנסיבית. זה האחרון תלוי ביטוי נפרד של תת-יחידות α ו β, ואחריו ערבוב. למרות requi זהמיל קצת יותר מאמץ מאשר Coexpression, זה בהחלט פיצו על ידי היכולת לזהות ביניים כי הם הפסידו במהלך Coexpression. יחד, שתי שיטות יכולות לספק תמונה שלמה יותר של הרכבה הפרוטאזום.

<table><tbody><tr><td>Acrylamide (40%) solution</td><td>Biorad</td><td>1610104</td><td>Unpolymerized acrylamide is a neurotoxin. Wear proper protective gear</td></tr><tr><td>Amicon ultra 0.5 mL centrifugal filters</td><td>EMDMillipore</td><td>UFC501024</td><td></td></tr><tr><td>Ammonium Persulfate</td><td>Sigma</td><td>A3678</td><td></td></tr><tr><td>ATP</td><td>Sigma</td><td>A7699</td><td></td></tr><tr><td>BCA assay kit</td><td>Pierce</td><td>23225</td><td></td></tr><tr><td>Bisacrylamide (2%) solution</td><td>Biorad</td><td>1610142</td><td></td></tr><tr><td>Bromophenol blue</td><td>Sigma</td><td>B8026</td><td></td></tr><tr><td>DNaseI</td><td>Sigma</td><td>DN25</td><td></td></tr><tr><td>Dithiothreitol (DTT)</td><td>Thermo Fisher</td><td>BP172</td><td></td></tr><tr><td>&lt;em&gt;E. coli&lt;/em&gt; BL21 competent cells</td><td>EMD Millipore</td><td>69450</td><td></td></tr><tr><td>GelCode Blue</td><td>Thermo Fisher</td><td>24592</td><td>Colloidal coomassie stain reagent for gels</td></tr><tr><td>Gel doc EZ system</td><td>Biorad</td><td>1708270</td><td>Gel documentation system</td></tr><tr><td>Gel releasers</td><td>Biorad</td><td>1653320</td><td>Wedge shaped plastic used to separate gel plates; useful for spreading liquid.</td></tr><tr><td>Glass rod</td><td>Thermo Fisher</td><td>11-380B</td><td></td></tr><tr><td>Glycerol</td><td>Sigma</td><td>49767</td><td></td></tr><tr><td>Glycine</td><td>Thermo Fisher</td><td>BP3865</td><td></td></tr><tr><td>Hamilton syringe</td><td>Thermo Fisher</td><td>14-813-38</td><td>Glass syringe for loading gels</td></tr><tr><td>HEPES</td><td>US Biologicals</td><td>H2010</td><td></td></tr><tr><td>HMW Native calibration kit</td><td>GE Healthcare</td><td>170445-01</td><td>High molecular weight protein standards</td></tr><tr><td>Hoefer SG30</td><td>Thermo Fisher</td><td>03-500-277</td><td>Gradient maker</td></tr><tr><td>Imidazole</td><td>US Biologicals</td><td>280671</td><td></td></tr><tr><td>IPTG</td><td>US Biologicals</td><td>I8500</td><td>For induction of protein expression</td></tr><tr><td>Isopropanol</td><td>Thermo Fisher</td><td>BP26181</td><td></td></tr><tr><td>Kanamycin sulfate</td><td>US Biologicals</td><td>K0010</td><td></td></tr><tr><td>Lysozyme</td><td>Sigma</td><td>L6876</td><td></td></tr><tr><td>MgCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Fluka analytical</td><td>630680</td><td></td></tr><tr><td>Mini Protean Tetra Cell</td><td>Biorad</td><td>1658002EDU</td><td>Gel electrophoresis apparatus</td></tr><tr><td>NaCl</td><td>Thermo Fisher</td><td>S640-3</td><td></td></tr><tr><td>NaOH</td><td>Thermo Fisher</td><td>S318-1</td><td></td></tr><tr><td>Pefabloc SC</td><td>Roche</td><td>11429876001</td><td>Protease inhibitor</td></tr><tr><td>pET42</td><td>EMD Millipore</td><td>70562</td><td>Expression plasmid</td></tr><tr><td>Precision plus all blue standard</td><td>Biorad</td><td>1610373</td><td>Molecular protein standard for SDS-PAGE</td></tr><tr><td>Quickchange mutagenesis kit</td><td>Agilent technologies</td><td>200521</td><td></td></tr><tr><td>Sodium Dodecyl Sulfate (SDS)</td><td>Thermo Fisher</td><td>BP166</td><td></td></tr><tr><td>Suc-LLVY-AMC</td><td>Enzo lifesciences</td><td>BML P802-0005</td><td>Fluorogenic substrate</td></tr><tr><td>Talon Metal Affinity Resin</td><td>Clontech</td><td>635502</td><td>Immobilized Cobalt Affinity Resin</td></tr><tr><td>TEMED</td><td>Sigma</td><td>T7024</td><td></td></tr><tr><td>Tris</td><td>US Biologicals</td><td>T8600</td><td></td></tr><tr><td>Triton-X100</td><td>Sigma</td><td>93426</td><td></td></tr><tr><td>Tryptone</td><td>Bacto BD</td><td>211699</td><td></td></tr><tr><td>UV sample tray</td><td>Biorad</td><td>1708271</td><td>For UV imaging of gels</td></tr><tr><td>Yeast extract</td><td>Bacto BD</td><td>212720</td><td></td></tr></tbody></table>

examining proteasome assembly with recombinant archaeal proteasomes and nondenaturing page:  the case for a combined approach

הפרוטאזומים נמצאים בכל תחומי החיים. הם מספקים את המסלול העיקרי של פירוק חלבונים תאיים אאוקריוטים, אם כי הרכבה שלהם אינה מובנת לחלוטין. כל פרוטאזומים מכילים חלקיק הליבה נשמרת מבנית (CP), או הפרוטאזום 20S, המכיל שתי טבעות למקטע β heptameric דחוקה בין שתי טבעות למקטע α heptameric. פרוטאזומים 20S Archaeal פשוטים לעומת בעלי הרכב לעמיתיהם איקריוטיים שלהם, בעודם חולקים מנגנון הרכבה משותף. כתוצאה מכך, פרוטאזומים 20S archaeal להמשיך להיות מודל חשוב להרכבת הפרוטאזום איקריוטיים. באופן ספציפי, ביטוי רקומביננטי של פרוטאזומים 20S archaeal בשילוב עם ג&#39;ל אלקטרופורזה polyacrylamide nondenaturing (עמוד) הניב תובנות חשובות רבות לתוך biogenesis הפרוטאזום. כאן, אנו דנים אמצעי לשפר את האסטרטגיה הרגילה של Coexpression של α הפרוטאזום archaeal ובטא יחידות משנה לפני nondenaturing עמוד. אנו מראים כי למרות מהירים ויעילים, גישת Coexpression לבדו יכולה לפספס ביניים הרכבת מפתח. במקרה של הפרוטאזום, Coexpression לא יכול לאפשר זיהוי של-הפרוטאזום וחצי, טבעת-α מלאה המכילה ביניים אחת β-טבעת אחד שלם. עם זאת, ביניים זו מזוהה בקלות באמצעות ערבוב lysate. אנו מציעים שילוב Coexpression עם ערבוב lysate מניב גישה יותר יסודית בניתוח הרכבה, עדיין נותר העבודה nonintensive. גישה זו עשויה להיות שימושית לחקר מתחמי multiprotein רקומביננטי אחרים.

הפרוטאזום הרכבה (איור 1) מתחילה כאשר יחידות משנת אלפא לשלב טבעות טופס 9. אפשר להדגים זאת כאשר יחידות משנה אלפא מן archaeon Methanococcus maripaludis S2 מבוטאים החיידק כמו C- סופני hexahistidine מתויג (מתויג שלו) נגזרים (טבלה 1). כאשר α-שלו רקומביננטי החלבון היה ?...

Watch this Scientific Journal Video about Examining Proteasome Assembly with Recombinant Archaeal Proteasomes and Nondenaturing PAGE:  The Case for a Combined Approach at JoVE.com

Examining Proteasome Assembly with Recombinant Archaeal Proteasomes and Nondenaturing PAGE:  The Case for a Combined Approach

פרוטוקול זה משתמש בשני Coexpression למקטע ו למקטע postlysis ערבוב לבדיקה יסודית יותר של הרכבת הפרוטאזום רקומביננטי.

בחינת העצרת הפרוטאזום עם רקומביננטי Archaeal הפרוטאזומים ועמוד Nondenaturing: The Case for גישה משולבת

Proteasomes are found in all domains of life. They provide the major route of intracellular protein degradation in eukaryotes, though their assembly is ...

examining-proteasome-assembly-with-recombinant-archaeal-proteasomes

Research

JoVE Journal

Biochemistry

6.6K Views.  Indiana University-Purdue University, Indianapolis (IUPUI).  פרוטוקול זה משתמש בשני Coexpression למקטע ו למקטע postlysis ערבוב לבדיקה יסודית יותר של הרכבת הפרוטאזום רקומביננטי. 

Video: Examining Proteasome Assembly with Recombinant Archaeal Proteasomes and Nondenaturing PAGE:  The Case for a Combined Approach

Directed Protein Packaging within Outer Membrane Vesicles from Escherichia coli: Design, Production and Purification

An increasing interest in applying synthetic biology techniques to program outer membrane vesicles (OMV) are leading to some very interesting and unique applications for OMV where traditional nanoparticles are proving too difficult to synthesize. To date, all Gram-negative bacteria have been shown to produce OMV demonstrating packaging of a variety of cargo that includes small molecules, peptides, proteins and genetic material. Based on their diverse cargo, OMV are implicated in many biological processes ranging from cell-cell communication to gene transfer and delivery of virulence factors depending upon which bacteria are producing the OMV. Only recently have bacterial OMV become accessible for use across a wide range of applications through the development of techniques to control and direct packaging of recombinant proteins into OMV. This protocol describes a method for the production, purification, and use of enzyme packaged OMV providing for improved overall production of recombinant enzyme, increased vesiculation, and enhanced enzyme stability. Successful utilization of this protocol will result in the creation of a bacterial strain that simultaneously produces a recombinant protein and directs it for OMV encapsulation through creating a synthetic linkage between the recombinant protein and an outer membrane anchor protein. This protocol also details methods for isolating OMV from bacterial cultures as well as proper handling techniques and things to consider when adapting this protocol for use for other unique applications such as: pharmaceutical drug delivery, medical diagnostics, and environmental remediation.

Directed Protein Packaging within Outer Membrane Vesicles from Escherichia coli: Design, Production and Purification

A protocol for the production, purification, and use of enzyme packaged outer membrane vesicles (OMV) providing for enhanced enzyme stability for implementation across diverse applications is presented.

אריזת חלבון בימוי בתוך החיצון ממברנה השלפוחית ​​מ<em&gt; Coli Escherichia</em&gt;: עיצוב, ייצור טיהור

An increasing interest in applying synthetic biology techniques to program outer membrane vesicles (OMV) are leading to some very interesting and unique ...

Online Size-exclusion and Ion-exchange Chromatography on a SAXS Beamline

Biological small angle X-ray scattering (BioSAXS) is a powerful technique in molecular and structural biology used to determine solution structure, particle size and shape, and surface-to-volume ratio of macromolecules. The technique is applicable to a very wide variety of solution conditions spanning a broad range of concentrations, pH values, ionic strengths, temperatures, additives, etc., but the sample is required to be monodisperse. This caveat led to the implementation of liquid chromatography systems on SAXS beamlines. Here, we describe the upstream integration of size-exclusion (SEC) and ion-exchange chromatography (IEC) on a beamline, different methods for optimal background subtraction, and data reduction. As an example, we describe how we use SEC- and IEC-SAXS on a fragment of the essential vaccinia virus protein D5, consisting of a D5N helicase domain. We determine its overall shape and molecular weight, showing the hexameric structure of the protein.

The determination of the solution structure of a protein by small angle X-ray scattering (SAXS) requires monodisperse samples. Here, we present two possibilities to ensure minimal delays between sample preparation and data acquisition: online size-exclusion chromatography (SEC) and online ion-exchange chromatography (IEC).

גודל הדרה מקוונת ו כרומטוגרפיה יון-חליפין על beamline SAXS

Biological small angle X-ray scattering (BioSAXS) is a powerful technique in molecular and structural biology used to determine solution structure, ...

Multimer-PAGE: A Method for Capturing and Resolving Protein Complexes in Biological Samples

There are many well-developed methods for purifying and studying single proteins and peptides. However, most cellular functions are carried out by networks of interacting protein complexes, which are often difficult to investigate because their binding is non-covalent and easily perturbed by purification techniques. This work describes a method of stabilizing and separating native protein complexes from unmodified tissue using two-dimensional polyacrylamide gel electrophoresis. Tissue lysate is loaded onto a non-denaturing blue-native polyacrylamide gel, then an electric current is applied until the protein migrates a short distance into the gel. The gel strip containing the migrated protein is then excised and incubated with the amine-reactive cross-linking reagent dithiobis(succinimidyl propionate), which covalently stabilizes protein complexes. The gel strip containing cross-linked complexes is then cast into a sodium dodecyl sulfate polyacrylamide gel, and the complexes are separated completely. The method relies on techniques and materials familiar to most molecular biologists, meaning it is inexpensive and easy to learn. While it is limited in its ability to adequately separate extremely large complexes, and has not been universally successful, the method was able to capture a wide variety of well-studied complexes, and is likely applicable to many systems of interest.

A method for stabilizing and separating native protein complexes from unmodified tissue lysate using an amine-reactive protein cross-linker coupled to a novel two-dimensional polyacrylamide gel electrophoresis (PAGE) system is presented.

Multimer-עמוד: שיטה לכידת וחלבון פתרון מתחמי דגימות ביולוגיות

There are many well-developed methods for purifying and studying single proteins and peptides. However, most cellular functions are carried out by ...

Reconstitution of Nucleosomes with Differentially Isotope-labeled Sister Histones

Asymmetrically modified nucleosomes contain the two copies of a histone (sister histones) decorated with distinct sets of Post-translational Modifications (PTMs). They are newly identified species with unknown means of establishment and functional implications. Current analytical methods are inadequate to detect the copy-specific occurrence of PTMs on the nucleosomal sister histones. This protocol presents a biochemical method for the in vitro reconstitution of nucleosomes containing differentially isotope-labeled sister histones. The generated complex can be also asymmetrically modified, after including a premodified histone pool during refolding of histone subcomplexes. These asymmetric nucleosome preparations can be readily reacted with histone-modifying enzymes to study modification cross-talk mechanisms imposed by the asymmetrically pre-incorporated PTM using nuclear magnetic resonance (NMR) spectroscopy. Particularly, the modification reactions in real-time can be mapped independently on the two sister histones by performing different types of NMR correlation experiments, tailored for the respective isotope type. This methodology provides the means to study crosstalk mechanisms that contribute to the formation and propagation of asymmetric PTM patterns on nucleosomal complexes.

This protocol describes the reconstitution of nucleosomes containing differentially isotope-labeled sister histones. At the same time, asymmetrically post-translationally modified nucleosomes can be generated after using a premodified histone copy. These preparations can be readily used to study modification crosstalk mechanisms, simultaneously on both sister histones, by using high-resolution NMR spectroscopy.

כינון של נוקלאוזום עם דיפרנציאלי איזוטופ שכותרתו היסטונים אחות

Asymmetrically modified nucleosomes contain the two copies of a histone (sister histones) decorated with distinct sets of Post-translational Modifications ...

Combining X-Ray Crystallography with Small Angle X-Ray Scattering to Model Unstructured Regions of Nsa1 from S. Cerevisiae

Determination of the full-length structure of ribosome assembly factor Nsa1 from Saccharomyces cerevisiae (S. cerevisiae) is challenging because of the disordered and protease labile C-terminus of the protein. This manuscript describes the methods to purify recombinant Nsa1 from S. cerevisiae for structural analysis by both X-ray crystallography and SAXS. X-ray crystallography was utilized to solve the structure of the well-ordered N-terminal WD40 domain of Nsa1, and then SAXS was used to resolve the structure of the C-terminus of Nsa1 in solution. Solution scattering data was collected from full-length Nsa1 in solution. The theoretical scattering amplitudes were calculated from the high-resolution crystal structure of the WD40 domain, and then a combination of rigid body and ab initio modeling revealed the C-terminus of Nsa1. Through this hybrid approach the quaternary structure of the entire protein was reconstructed. The methods presented here should be generally applicable for the hybrid structural determination of other proteins composed of a mix of structured and unstructured domains.

Combining X-Ray Crystallography with Small Angle X-Ray Scattering to Model Unstructured Regions of Nsa1 from S. Cerevisiae

This method describes the cloning, expression, and purification of recombinant Nsa1 for structural determination by X-ray crystallography and small-angle X-ray scattering (SAXS), and is applicable for the hybrid structural analysis of other proteins containing both ordered and disordered domains.

שילוב קריסטלוגרפיה באמצעות קרני רנטגן עם זווית קטנה פיזור קרני רנטגן ליצור מודל לא מובנה מחוזות Nsa1 של Cerevisiae ס

Determination of the full-length structure of ribosome assembly factor Nsa1 from Saccharomyces cerevisiae (S. cerevisiae) is challenging because of the ...

Use of Recombinant Fusion Proteins in a Fluorescent Protease Assay Platform and Their In-gel Renaturation

Proteases are intensively studied enzymes due to their essential roles in several biological pathways of living organisms and in pathogenesis; therefore, they are important drug targets. We have developed a magnetic-agarose-bead-based assay platform for the investigation of proteolytic activity, which is based on the use of recombinant fusion protein substrates. In order to demonstrate the use of this assay system, a protocol is presented on the example of human immunodeficiency virus type 1 (HIV-1) protease. The introduced assay platform can be utilized efficiently in the biochemical characterization of proteases, including enzyme activity measurements in mutagenesis, kinetic, inhibition, or specificity studies, and it may be suitable for high-throughput substrate screening or may be adapted to other proteolytic enzymes.
In this assay system, the applied substrates contain N-terminal hexahistidine (His6) and maltose-binding protein (MBP) tags, cleavage sites for tobacco etch virus (TEV) and HIV-1 proteases, and a C-terminal fluorescent protein. The substrates can be efficiently produced in Escherichia coli cells and easily purified using nickel (Ni)-chelate-coated beads. During the assay, the proteolytic cleavage of bead-attached substrates leads to the release of fluorescent cleavage fragments, which can be measured by fluorimetry. Additionally, cleavage reactions can be analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). A protocol for the in-gel renaturation of assay components is also described, as partial renaturation of fluorescent proteins enables their detection based on molecular weight and fluorescence.

Here, we present the detailed procedure of a recently developed protease assay platform utilizing N-terminal hexahistidine/maltose-binding protein and fluorescent protein-fused recombinant substrates attached to the surface of nickel-nitrilotriacetic acid magnetic agarose beads. A subsequent in-gel analysis of the assay samples separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis is also presented.

השימוש פיוז'ן רקומביננטי חלבונים פלטפורמה Assay פרוטאז פלורסנט, Renaturation שלהם בג'ל

Proteases are intensively studied enzymes due to their essential roles in several biological pathways of living organisms and in pathogenesis; therefore, ...

OaAEP1-Mediated Enzymatic Synthesis and Immobilization of Polymerized Protein for Single-Molecule Force Spectroscopy

Chemical and bio-conjugation techniques have been developed rapidly in recent years and allow the building of protein polymers. However, a controlled protein polymerization process is always a challenge. Here, we have developed an enzymatic methodology for constructing polymerized protein step by step in a rationally-controlled sequence. In this method, the C-terminus of a protein monomer is NGL for protein conjugation using OaAEP1 (Oldenlandia affinis asparaginyl endopeptidases) 1) while the N-terminus was a cleavable TEV (tobacco etch virus) cleavage site plus an L (ENLYFQ/GL) for temporary N-terminal protecting. Consequently, OaAEP1 was able to add only one protein monomer at a time, and then the TEV protease cleaved the N-terminus between Q and G to expose the NH2-Gly-Leu. Then the unit is ready for next OaAEP1 ligation. The engineered polyprotein is examined by unfolding individual protein domain using atomic force microscopy-based single-molecule force spectroscopy (AFM-SMFS). Therefore, this study provides a useful strategy for polyprotein engineering and immobilization.

Here, we present a protocol to conjugate protein monomer by enzymes forming protein polymer with a controlled sequence and immobilize it on the surface for single-molecule force spectroscopy studies.

OaAEP1-תיווך אנזימטית והשתק של חלבון פלסטריזציה לספקטרוסקופיית מולקולות יחיד

Chemical and bio-conjugation techniques have been developed rapidly in recent years and allow the building of protein polymers. However, a controlled ...

High-throughput Purification of Affinity-tagged Recombinant Proteins

X-ray crystallography is the method of choice for obtaining a detailed view of the structure of proteins. Such studies need to be complemented by further biochemical analyses to obtain detailed insights into structure/function relationships. Advances in oligonucleotide- and gene synthesis technology make large-scale mutagenesis strategies increasingly feasible, including the substitution of target residues by all 19 other amino acids. Gain- or loss-of-function phenotypes then allow systematic conclusions to be drawn, such as the contribution of particular residues to catalytic activity, protein stability and/or protein-protein interaction specificity.
In order to attribute the different phenotypes to the nature of the mutation - rather than to fluctuating experimental conditions - it is vital to purify and analyse the proteins in a controlled and reproducible manner. High-throughput strategies and the automation of manual protocols on robotic liquid-handling platforms have created opportunities to perform such complex molecular biological procedures with little human intervention and minimal error rates1-5.
Here, we present a general method for the purification of His-tagged recombinant proteins in a high-throughput manner. In a recent study, we applied this method to a detailed structure-function investigation of TFIIB, a component of the basal transcription machinery. TFIIB is indispensable for promoter-directed transcription in vitro and is essential for the recruitment of RNA polymerase into a preinitiation complex6-8. TFIIB contains a flexible linker domain that penetrates the active site cleft of RNA polymerase9-11. This linker domain confers two biochemically quantifiable activities on TFIIB, namely (i) the stimulation of the catalytic activity during the 'abortive' stage of transcript initiation, and (ii) an additional contribution to the specific recruitment of RNA polymerase into the preinitiation complex4,5,12 . We exploited the high-throughput purification method to generate single, double and triple substitution and deletions mutations within the TFIIB linker and to subsequently analyse them in functional assays for their stimulation effect on the catalytic activity of RNA polymerase4. Altogether, we generated, purified and analysed 381 mutants - a task which would have been time-consuming and laborious to perform manually. We produced and assayed the proteins in multiplicates which allowed us to appreciate any experimental variations and gave us a clear idea of the reproducibility of our results.
This method serves as a generic protocol for the purification of His-tagged proteins and has been successfully used to purify other recombinant proteins. It is currently optimised for the purification of 24 proteins but can be adapted to purify up to 96 proteins.

We describe a method for the affinity-tagged purification of recombinant proteins using liquid-handling robotics. This method is generally applicable to the small-scale purification of soluble His-tagged proteins in a high-throughput format.

טיהור תפוקה גבוהה של חלבונים רקומביננטי זיקה מתויגת-

X-ray crystallography is the method of choice for obtaining a detailed view of the structure of proteins. Such studies need to be complemented by further ...

Biology

Residue-Specific Exchange of Proline by Proline Analogs in Fluorescent Proteins: How "Molecular Surgery" of the Backbone Affects Folding and Stability

Replacement of proline (Pro) residues in proteins by the traditional site-directed mutagenesis by any of the remaining 19 canonical amino acids is often detrimental to protein folding and, in particular, chromophore maturation in green fluorescent proteins and related variants. A reasonable alternative is to manipulate the translation of the protein so that all Pro residues are replaced residue-specifically by analogs, a method known as selective pressure incorporation (SPI). The built-in chemical modifications can be used as a kind of &#34;molecular surgery&#34; to finely dissect measurable changes or even rationally manipulate different protein properties. Here, the study demonstrates the usefulness of the SPI method to study the role of prolines in the organization of the typical &#946;-barrel structure of spectral variants of the green fluorescent protein (GFP) with 10-15 prolines in their sequence: enhanced green fluorescent protein (EGFP), NowGFP, and KillerOrange. Pro residues are present in connecting sections between individual &#946;-strands and constitute the closing lids of the barrel scaffold, thus being responsible for insulation of the chromophore from water, i.e., fluorescence properties. Selective pressure incorporation experiments with (4R)-fluoroproline (R-Flp), (4S)-fluoroproline (S-Flp), 4,4-difluoroproline (Dfp), and 3,4-dehydroproline (Dhp) were performed using a proline-auxotrophic E. coli strain as expression host. We found that fluorescent proteins with S-Flp and Dhp are active (i.e., fluorescent), while the other two analogs (Dfp and R-Flp) produced dysfunctional, misfolded proteins. Inspection of UV-Vis absorption and fluorescence emission profiles showed few characteristic alterations in the proteins containing Pro analogs. Examination of the folding kinetic profiles in EGFP variants showed an accelerated refolding process in the presence of S-Flp, while the process was similar to wild-type in the protein containing Dhp. This study showcases the capacity of the SPI method to produce subtle modifications of protein residues at an atomic level (&#34;molecular surgery&#34;), which can be adopted for the study of other proteins of interest. It illustrates the outcomes of proline replacements with close chemical analogs on the folding and spectroscopic properties in the class of &#946;-barrel fluorescent proteins.

To overcome the limitations of classical site-directed mutagenesis, proline analogs with specific modifications were incorporated into several fluorescent proteins. We show how the replacement of hydrogen by fluorine or of the single by double bonds in proline residues ("molecular surgery") affects fundamental protein properties, including their folding and interaction with light.

חילופי פרולין ספציפיים לשאריות על ידי אנלוגים פרולין בחלבונים פלואורסצנטיים: כיצד "ניתוח מולקולרי" של עמוד השדרה משפיע על קיפול ויציבות

Replacement of proline (Pro) residues in proteins by the traditional site-directed mutagenesis by any of the remaining 19 canonical amino acids is often ...

Bioengineering

The Proteasome Structure

The ubiquitin-proteasome pathway is a well-known mechanism utilized by eukaryotic cells to remove cytoplasmic proteins that are misfolded, damaged, or no longer needed. In this pathway, the protein that needs to be eliminated undergoes a process called ubiquitination, where a chain of ubiquitin molecules is attached to the 48th lysine residue of the target protein. This ubiquitin modification helps the proteasome distinguish between a target protein and a healthy protein.
The proteasome is an ATP-driven, large multi-subunit protease (26S) found in the nucleus and cytoplasm of all eukaryotes. The 26S proteasome comprises a central hollow cylindrical core (the 20S) and ring-shaped protein caps (19S). The 20S core particle is barrel-shaped, containing stacked rings with proteolytic active sites lined inside. The 19S caps act as gatekeepers and regulatory units of the proteasome. They are present at one or both ends of the core.
Caps have ubiquitin receptors, ubiquitin hydrolase, and unfoldase, each with a unique function. The ubiquitin receptor recognizes and allows ubiquitin-tagged proteins into the core, the hydrolase deubiquitinase cleaves ubiquitin from the substrate, and the unfoldase protein unfolds target proteins as they enter the protein core.
The hexameric unfoldases create a ring structure in the proteasome cap and belong to a large class of AAA proteins. Their mechanistic action is similar to other ATP-dependent helicases that cause DNA unwinding. The ATP-bound unfoldase ring binds a folded target protein held in place by its ubiquitin tag at the proteasomal cap. Next, a conformational change, driven by ATP hydrolysis, pulls the target protein substrate into the 20S core and strains the ring structure. As a result, when a protein is subjected to pulling forces, it can undergo two possible outcomes&#8212; it may either undergo partial unfolding and enter the cylindrical core, or it can retain its structure while partially retracting.
Highly stable substrates generally require hundreds of cycles of ATP hydrolysis before being pulled through the AAA ring. Once de-ubiquitylated and unfolded, the target protein is exposed to proteases inside the proteasomal core for digestion. In contrast to free cellular proteases, which quickly dissociate after their action on the target protein, the proteasome core holds the target protein until it is fully digested into smaller peptides. These small peptides are then released into the cytosol for further processing.&#160;

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The ubiquitin-proteasome pathway is a well-known mechanism utilized by eukaryotic cells to remove cytoplasmic proteins that are misfolded, damaged, or no ...

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בחינת העצרת הפרוטאזום עם רקומביננטי Archaeal הפרוטאזומים ועמוד Nondenaturing: The Case for גישה משולבת

Summary

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Chapters in this video

אריזת חלבון בימוי בתוך החיצון ממברנה השלפוחית ​​מ

גודל הדרה מקוונת ו כרומטוגרפיה יון-חליפין על beamline SAXS

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כינון של נוקלאוזום עם דיפרנציאלי איזוטופ שכותרתו היסטונים אחות

שילוב קריסטלוגרפיה באמצעות קרני רנטגן עם זווית קטנה פיזור קרני רנטגן ליצור מודל לא מובנה מחוזות Nsa1 של Cerevisiae ס

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טיהור תפוקה גבוהה של חלבונים רקומביננטי זיקה מתויגת-

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