이 작품은 건강 부여 GM086822 호프, 심장 연구소 기초 과학 수상하고, MJ 머독 자선 신탁 대학 과학 연구 프로그램의 국립 연구소에 의해 투자되었다. 전기 영동의 시약을 선택하고 이미지 분석 소프트웨어는 넉넉한 바이오 방사선에 의해 제공되었다.

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(30), e1293-e1293 (2009).</li><li>Murphy, P. J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualization+and+mechanism+of+assembly+of+a+glucocorticoid+receptor&amp;bull;Hsp70+complex+that+is+primed+for+subsequent+Hsp90-dependent+opening+of+the+steroid+binding+cleft.">Visualization and mechanism of assembly of a glucocorticoid receptor&amp;bull;Hsp70 complex that is primed for subsequent Hsp90-dependent opening of the steroid binding cleft.</a> J. Biol. Chem. 278, 34764-34773 (2003).</li><li>Schrum, A. 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전기 영동의 시약을 선택하고 이미지 분석 소프트웨어는 바이오 방사선에 의해 제공되었다.

위에서 설명한 분석은 hsp90/hsp70-based 보호자 기계의 보호자 역할 행동뿐만 아니라, GR 스테로이드 바인딩에 영향을 미치는 조건의 수많은 테스트를 적용할 수 있습니다. 그것은 이전에 보고된 방법 4,8,9,17의 수정이며 전기 영동 기술의 최근 발전을 활용하고 광범위한 연구 커뮤니티에 액세스할 수 있도록 설계되었습니다. 관심의 추가 cofactors이나 단백질은 GR에 추가할 수 있습니다 스테로?...

<table border="1"><tbody><tr><th> 시약의 이름 </th><th> 회사 </th><th> 카탈로그 번호 </th><th> 댓글 </th></tr><tr><td> Sf9 곤충 세포 </td><td> Invitrogen </td><td></td><td> 재조합 마우스 GR의 baculovirus 발현을위한 </td></tr><tr><td> L929 세포 </td><td> ATCC </td><td> CRL - 2173 </td><td> 내생 마우스 GR cytosol 준비 (baculovirus 발현에 대안)에 대한 </td></tr><tr><td> FiGR 하이 브리 도마 세포 </td><td> ATCC </td><td> CRL - 2173 </td><td> (정화 BuGR2 항체로 대체) 안티 - GR 단클론 항체를 포함하는 복수를 준비 </td></tr><tr><td> 완료 - 미니 테아제 억제제, EDTA (에틸렌 다이아 민 테트라 초산) - 무료 </td><td> 호슈 응용 과학 </td><td> 11836170001 </td><td></td></tr><tr><td> 단백질 A - 세파 로스 </td><td> 시그마 - 올드 리치 </td><td> P3391 </td><td></td></tr><tr><td> 토끼 reticulocyte lysate </td><td> 그린 헥타르 (오레곤, WI) </td><td></td><td> 내생 hsp90/hsp70 보호자 기계 풍부한 소스 </td></tr><tr><td> 크레아틴 phosphokinase </td><td> 시그마 - 올드 리치 </td><td> C3755 </td><td> ATP - 재생 시스템 </td></tr><tr><td> phosphocreatine </td><td> 시그마 - 올드 리치 </td><td> P7936 </td><td> ATP - 재생 시스템 </td></tr><tr><td> 안티 GR 마우스 단클론 항체 (BuGR2) </td><td> 피어스 / 써모 과학 </td><td> MA1 - 510 </td><td> GR immunoadsorption 사용과 서양 blotting에서와 같이 기본 항체 </td></tr><tr><td> 안티 hsp90 마우스 단클론 항체 (AC88) </td><td> 엔조 생명 과학 </td><td> ADI - SPA - 830 </td><td> 서쪽은 blotting의 기본 항체로 사용 </td></tr><tr><td> 안티 hsp70 마우스 단클론 항체 (N27F3 - 4) </td><td> 엔조 생명 과학 </td><td> ADI - SPA - 820 </td><td> 서쪽은 blotting의 기본 항체로 사용 </td></tr><tr><td> 마우스 혈청에서 IgG </td><td> 시그마 - 올드 리치 </td><td> 038K7690 </td><td> immunoadsorption의 부정적인 제어를위한 nonimmune 항체로 사용 </td></tr><tr><td> 안티 - 마우스 IgG - 퍼옥시데이즈의 활용 </td><td> 시그마 - 올드 리치 </td><td> A4416 </td><td> 서쪽은 blotting에서 보조 항체로 사용 </td></tr><tr><td> Experion microfluidic 전기 영동 시스템 </td><td> 바이오 방사선 </td><td> 7,007,001 </td><td> 종래의 SDS - PAGE로 대체 </td></tr><tr><td> [1,2,4,6,7 - 3 H] dexamethasone </td><td> PerkinElmer </td><td> NET1192001MC </td><td> 안전주의 사항을 따르십시오 </td></tr></tbody></table>

biochemical reconstitution of steroid receptor&#x2022;hsp90 protein  complexes and reactivation of ligand binding

Hsp90은 세포 자전거에 관련된 전사 인자 (예 : 핵 수용체, p53의)와 단백질 kinases (예 : SRC, RAF, Akt 키나제)를 포함한 150 개 이상의 진핵세포 신호 단백질을 조절하는 것으로 판명되었습니다 순수하고 매우 풍부한 분자 보호자 역할 단백질입니다 tumorigenesis, apoptosis, 여러 진핵세포 신호 경로 1,2. hsp90 이러한 다양한 &#39;고객&#39;단백질의 스테로이드 수용체의 조립 • hsp90 단지는 (그림 1) 정의 가장됩니다. 우리는 여기서 적응 글루코 코르티 코 이드 수용체 (GR) immunoprecipitation 분석과 쉽게 진핵세포 hsp90 기능 활동, 바인딩 hsp90 - 중재 스테로이드 수용체 리간드 및 분자 보호자 역할 cofactor 요구 사항을 탐사하는 데 사용할 수 있습니다 체외 GR에 • hsp90 reconstitution 방법을 제시한다. 예를 들어,이 분석은 hsp90 cofactor 요구 사항 및 reconstitution 과정 외인성 화합물을 추가의 효과를 테스트하는 데 사용할 수 있습니다. 수용체는 스테로이드 3 액세스할 수 있습니다 분열 오픈 리간드 바인딩을 hsp90에 바인딩해야하기 때문에 GR은 hsp90를 공부에 특히 유용 시스템되었습니다. 내생, unliganded GR은 noncovalently hsp90에 바인딩 포유 동물 세포의 세포질에 존재합니다. 이름으로 내생 GR • hsp90 heterocomplex에서 발견, 갈라진 GR의 리간드 바인딩은 개방적이고 구속력 스테로이드 가능합니다. hsp90 GR에서 dissociates 또는 그 기능이 저해되면 수용체는 스테로이드 구속 수 있으며, 스테로이드 구속력이 활동을하기 전에 GR • hsp90 heterocomplex의 reconstitution을 필요로 4를 복원하는 경우. GR은 단클론 항체를 사용하여 셀 cytosol에서 immunoprecipitated, 그리고 서양 얼룩에 의해 assayed 수있는 등 hsp90와 같은 단백질은 GR에 complexed 수 있습니다. immunoprecipitated GR의 스테로이드 결합 활동은 [3 H] 스테로이드와 immunopellet을 잠복기에 의해 결정하실 수 있습니다. 이전 실험 분열 GR의 리간드 바인딩의 hsp90 - 중재 열기를 보이고있다하면 진핵 세포 생존을위한 hsp70, 두 번째 분자 보호자도 중요합니다. hsp90 및 hsp70의 생화 학적 활동이 공동 보호자 역할 단백질의 홉, hsp40, 그리고 p23 5 촉매 있습니다. hsp90, hsp70, 타, 그리고 hsp40를 포함하는 multiprotein의 보호자 기계는 진핵 세포 세포질에 endogenously 존재하고 있으며, reticulocyte lysate는 보호자 풍부한 단백질 소스 여섯를 제공합니다. 표시 방법에서 GR은​​ 셀 cytosol에서 immunoadsorbed하고 온화한 소금 조건을 사용하여 내생 hsp90/hsp70 보호자 기계 찟겼어. 소금 막았죠 GR은 다음 GR의 reconstitution 결과 reticulocyte lysate, ATP, 그리고 K +와 incubated입니다 스테로이드 바인딩 활동 7 • hsp90 heterocomplex 및 재활 성화. 이 방법은 hsp90 - 중재 스테로이드 결합 8-11 그들의 기능 중요성을 결정하기 위해 다양한 보호자 역할 cofactors, 소설의 단백질, 그리고 실험 hsp90 또는 GR 억제제의 효과를 테스트하기 위해 이용하실 수 있습니다.

Watch this Scientific Journal Video about Biochemical Reconstitution of Steroid Receptorâ€¢Hsp90 Protein  Complexes and Reactivation of Ligand Binding at JoVE.com

Biochemical Reconstitution of Steroid Receptor&amp;#x2022;Hsp90 Protein  Complexes and Reactivation of Ligand Binding

<em&gt; 체외에서</em• hsp90 단백질 단지 단백질과 세포 lysates을 정화의 기능 글루코 코르티 코 이드 수용체 (GR)를 준비&gt; 방법을 설명합니다. 방법은 소금 조직 및 단백질 복잡한 reconstitution 다음 재조합 GR의 immunoadsorption을 활용합니다. 잠재적인 방법을 응용 프로그램이 같은 cofactors와 버퍼 조건의 중요성이 논의됩니다.

스테로이드 수용체의 생화 학적 리간드의 Reconstitution • Hsp90 단백질 단지와 재가입 바인딩

Hsp90 is an essential and highly abundant molecular chaperone protein that has been found to regulate more than 150 eukaryotic signaling proteins, ...

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Biochemical Reconstitution of Steroid Receptor&#x2022;Hsp90 Protein  Complexes and Reactivation of Ligand Binding

16.3K 조회수.  Seattle University.  체외에서 방법을 설명합니다. 방법은 소금 조직 및 단백질 복잡한 reconstitution 다음 재조합 GR의 immunoadsorption을 활용합니다. 잠재적인 방법을 응용 프로그램이 같은 cofactors와 버퍼 조건의 중요성이 논의됩니다.

비디오: Biochemical Reconstitution of Steroid Receptor&#x2022;Hsp90 Protein  Complexes and Reactivation of Ligand Binding

Identification of protein complexes with quantitative proteomics in S. cerevisiae

Lipids are the building blocks of cellular membranes that function as barriers and in compartmentalization of cellular processes, and recently, as important intracellular signalling molecules. However, unlike proteins, lipids are small hydrophobic molecules that traffic primarily by poorly described nonvesicular routes, which are hypothesized to occur at membrane contact sites (MCSs). MCSs are regions where the endoplasmic reticulum (ER) makes direct physical contact with a partnering organelle, e.g., plasma membrane (PM). The ER portion of ER-PM MCSs is enriched in lipid-synthesizing enzymes, suggesting that lipid synthesis is directed to these sites and implying that MCSs are important for lipid traffic. Yeast is an ideal model to study ER-PM MCSs because of their abundance, with over 1000 contacts per cell, and their conserved nature in all eukaryotes. Uncovering the proteins that constitute MCSs is critical to understanding how lipids traffic is accomplished in cells, and how they act as signaling molecules. We have found that an ER called Scs2p localize to ER-PM MCSs and is important for their formation. We are focused on uncovering the molecular partners of Scs2p. Identification of protein complexes traditionally relies on first resolving purified protein samples by gel electrophoresis, followed by in-gel digestion of protein bands and analysis of peptides by mass spectrometry. This often limits the study to a small subset of proteins. Also, protein complexes are exposed to denaturing or non-physiological conditions during the procedure. To circumvent these problems, we have implemented a large-scale quantitative proteomics technique to extract unbiased and quantified data. We use stable isotope labeling with amino acids in cell culture (SILAC) to incorporate staple isotope nuclei in proteins in an untagged control strain. Equal volumes of tagged culture and untagged, SILAC-labeled culture are mixed together and lysed by grinding in liquid nitrogen. We then carry out an affinity purification procedure to pull down protein complexes. Finally, we precipitate the protein sample, which is ready for analysis by high-performance liquid chromatography/ tandem mass spectrometry. Most importantly, proteins in the control strain are labeled by the heavy isotope and will produce a mass/ charge shift that can be quantified against the unlabeled proteins in the bait strain. Therefore, contaminants, or unspecific binding can be easily eliminated. By using this approach, we have identified several novel proteins that localize to ER-PM MCSs. Here we present a detailed description of our approach. 

Here we describe a new quantitative proteomics technique for identifying protein complexes in Saccharomyces cerevisiae. In this study, we have used the SILAC method together with affinity purification followed by tandem mass spectrometry to identify with high specificity the binding partners of an ER protein, Scs2p.

S. cerevisiae의의 양적 proteomics와 단백질 단지의 식별

Lipids are the building blocks of cellular membranes that function as barriers and in compartmentalization of cellular processes, and recently, as ...

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생물학

Analyzing Large Protein Complexes by Structural Mass Spectrometry

Living cells control and regulate their biological processes through the coordinated action of a large number of proteins that assemble themselves into an array of dynamic, multi-protein complexes1. To gain a mechanistic understanding of the various cellular processes, it is crucial to determine the structure of such protein complexes, and reveal how their structural organization dictates their function. Many aspects of multi-protein complexes are, however, difficult to characterize, due to their heterogeneous nature, asymmetric structure, and dynamics. Therefore, new approaches are required for the study of the tertiary levels of protein organization.
One of the emerging structural biology tools for analyzing macromolecular complexes is mass spectrometry (MS)2-5. This method yields information on the complex protein composition, subunit stoichiometry, and structural topology. The power of MS derives from its high sensitivity and, as a consequence, low sample requirement, which enables examination of protein complexes expressed at endogenous levels. Another advantage is the speed of analysis, which allows monitoring of reactions in real time. Moreover, the technique can simultaneously measure the characteristics of separate populations co-existing in a mixture. 
Here, we describe a detailed protocol for the application of structural MS to the analysis of large protein assemblies. The procedure begins with the preparation of gold-coated capillaries for nanoflow electrospray ionization (nESI). It then continues with sample preparation, emphasizing the buffer conditions which should be compatible with nESI on the one hand, and enable to maintain complexes intact on the other. We then explain, step-by-step, how to optimize the experimental conditions for high mass measurements and acquire MS and tandem MS spectra. Finally, we chart the data processing and analyses that follow. Rather than attempting to characterize every aspect of protein assemblies, this protocol introduces basic MS procedures, enabling the performance of MS and MS/MS experiments on non-covalent complexes. Overall, our goal is to provide researchers unacquainted with the field of structural MS, with knowledge of the principal experimental tools.

Mass spectrometry has proven to be a valuable tool for analyzing large protein complexes. This method enables insights into the composition, stoichiometry and overall architecture of multi-subunit assemblies. Here, we describe, step-by-step, how to perform a structural mass spectrometry analysis, and characterize macromolecular structures.

구조 질량 분광법에 의해 큰 단백질 단지 분석

Living cells control and regulate their biological processes through the coordinated action of a large number of proteins that assemble themselves into an ...

Biochemical Titration of Glycogen In vitro

Glycogen is the main energetic polymer of glucose in vertebrate animals and plays a crucial role in whole body metabolism as well as in cellular metabolism. Many methods to detect glycogen already exist but only a few are quantitative. We describe here a method using the Abcam Glycogen assay kit, which is based on specific degradation of glycogen to glucose by glucoamylase. Glucose is then specifically oxidized to a product that reacts with the OxiRed probe to produce fluorescence. Titration is accurate, sensitive and can be achieved on cell extracts or tissue sections. However, in contrast to other techniques, it does not give information about the distribution of glycogen in the cell. As an example of this technique, we describe here the titration of glycogen in two cell lines, Chinese hamster lung fibroblast CCL39 and human colon carcinoma LS174, incubated in normoxia (21% O2) versus hypoxia (1% O2). We hypothesized that hypoxia is a signal that prepares cells to synthesize and store glycogen in order to survive1.

Biochemical Titration of Glycogen In vitro

We describe here an accurate, reproducible and convenient biochemical method for titration of glycogen in vitro. This technique uses the Abcam Glycogen assay kit and is based on successive hydrolysis of glycogen to glucose and glucose titration by fluorescence.

글리코겐의 생화학 적정<em&gt; 체외</em

Glycogen is the main energetic polymer of glucose in vertebrate animals and plays a crucial role in whole body metabolism as well as in cellular ...

Protein Purification-free Method of Binding Affinity Determination by Microscale Thermophoresis

Quantitative characterization of protein interactions is essential in practically any field of life sciences, particularly drug discovery. Most of currently available methods of KD determination require access to purified protein of interest, generation of which can be time-consuming and expensive. We have developed a protocol that allows for determination of binding affinity by microscale thermophoresis (MST) without purification of the target protein from cell lysates. The method involves overexpression of the GFP-fused protein and cell lysis in non-denaturing conditions. Application of the method to STAT3-GFP transiently expressed in HEK293 cells allowed to determine for the first time the affinity of the well-studied transcription factor to oligonucleotides with different sequences. The protocol is straightforward and can have a variety of application for studying interactions of proteins with small molecules, peptides, DNA, RNA, and proteins.

Microscale thermophoresis (MST) can be widely used for determination of binding affinity without purification of the target protein from cell lysates. The protocol involves overexpression of the GFP-fused protein, cell lysis in non-denaturing conditions, and detection of MST signal in the presence of varying concentrations of the ligand.

미세 규모 Thermophoresis에 의한 결합력 결정 단백질 정제 - 무료 방법

Quantitative characterization of protein  interactions is essential in practically any field of life sciences,  particularly drug discovery. Most of ...

Identification of Post-translational Modifications of Plant Protein Complexes

Plants adapt quickly to changing environments due to elaborate perception and signaling systems. During pathogen attack, plants rapidly respond to infection via the recruitment and activation of immune complexes. Activation of immune complexes is associated with post-translational modifications (PTMs) of proteins, such as phosphorylation, glycosylation, or ubiquitination. Understanding how these PTMs are choreographed will lead to a better understanding of how resistance is achieved.
Here we describe a protein purification method for nucleotide-binding leucine-rich repeat (NB-LRR)-interacting proteins and the subsequent identification of their post-translational modifications (PTMs). With small modifications, the protocol can be applied for the purification of other plant protein complexes. The method is based on the expression of an epitope-tagged version of the protein of interest, which is subsequently partially purified by immunoprecipitation and subjected to mass spectrometry for identification of interacting proteins and PTMs.
This protocol demonstrates that: i). Dynamic changes in PTMs such as phosphorylation can be detected by mass spectrometry; ii). It is important to have sufficient quantities of the protein of interest, and this can compensate for the lack of purity of the immunoprecipitate; iii). In order to detect PTMs of a protein of interest, this protein has to be immunoprecipitated to get a sufficient quantity of protein.

We describe here a protocol for the purification and characterization of plant protein complexes. We demonstrate that by immunoprecipitating a single protein within a complex, so we can identify its post-translational modifications and its interacting partners.

식물 단백질 복합체의 번역 후 변형의 확인

Plants adapt quickly to changing environments due to elaborate perception and signaling systems. During pathogen attack, plants rapidly respond to ...

AFM-based Mapping of the Elastic Properties of Cell Walls: at Tissue, Cellular, and Subcellular Resolutions

We describe a recently developed method to measure mechanical properties of the surfaces of plant tissues using atomic force microscopy (AFM) micro/nano-indentations, for a JPK&#160;AFM. Specifically, in this protocol we measure the apparent Young&#8217;s modulus of cell walls at subcellular resolutions across regions of up to 100 &#181;m&#160;x 100 &#181;m in floral meristems, hypocotyls, and roots. This requires careful preparation of the sample, the correct selection of micro-indenters and indentation depths. To account for cell wall properties only, measurements are performed in highly concentrated solutions of mannitol in order to plasmolyze the cells and thus remove the contribution of cell turgor pressure.
In contrast to other extant techniques, by using different indenters and indentation depths, this method allows simultaneous multiscale measurements, i.e. at subcellular resolutions and across hundreds of cells comprising a tissue. This means that it is now possible&#160;to spatially-temporally characterize the changes that take place in the mechanical properties of cell walls during development, enabling these changes to be correlated with growth and differentiation. This represents a key step to understand how coordinated microscopic cellular changes bring about macroscopic morphogenetic events.
However, several limitations remain: the method can only be used on fairly small samples (around 100 &#181;m in diameter) and only on external tissues; the method is sensitive to tissue topography; it measures only certain aspects of the tissue&#8217;s complex mechanical properties. The technique is being developed rapidly and it is likely that most of these limitations will be resolved in the near future.

We describe a method to map mechanical properties of plant tissues using an atomic force microscope (AFM). We focus on how to record mechanical changes that take place in cell walls during plant development at wide-field mesoscale, enabling these changes to be correlated with growth and morphogenesis.

셀 벽의 탄성 특성의 AFM 기반의 매핑 : 조직, 세포 및 세포 이하의 해상도에서

We describe a recently developed method to measure mechanical properties of the surfaces of plant tissues using atomic force microscopy (AFM) ...

Determination of Protein-ligand Interactions Using Differential Scanning Fluorimetry

A wide range of methods are currently available for determining the dissociation constant between a protein and interacting small molecules. However, most of these require access to specialist equipment, and often require a degree of expertise to effectively establish reliable experiments and analyze data. Differential scanning fluorimetry (DSF) is being increasingly used as a robust method for initial screening of proteins for interacting small molecules, either for identifying physiological partners or for hit discovery. This technique has the advantage that it requires only a PCR machine suitable for quantitative PCR, and so suitable instrumentation is available in most institutions; an excellent range of protocols are already available; and there are strong precedents in the literature for multiple uses of the method. Past work has proposed several means of calculating dissociation constants from DSF data, but these are mathematically demanding. Here, we demonstrate a method for estimating dissociation constants from a moderate amount of DSF experimental data. These data can typically be collected and analyzed within a single day. We demonstrate how different models can be used to fit data collected from simple binding events, and where cooperative binding or independent binding sites are present. Finally, we present an example of data analysis in a case where standard models do not apply. These methods are illustrated with data collected on commercially available control proteins, and two proteins from our research program. Overall, our method provides a straightforward way for researchers to rapidly gain further insight into protein-ligand interactions using DSF.

Differential scanning fluorimetry is a widely used method for screening libraries of small molecules for interactions with proteins. Here, we present a straightforward method to extend these analyses to provide an estimate of the dissociation constant between a small molecule and its protein partner.

시차 주사 Fluorimetry를 사용하여 단백질 - 리간드 상호 작용의 결정

A wide range of methods are currently available for determining the dissociation constant between a protein and interacting small molecules. However, most ...

In Vitro Reconstitution of Light-harvesting Complexes of Plants and Green Algae

In plants and green algae, light is captured by the light-harvesting complexes (LHCs), a family of integral membrane proteins that coordinate chlorophylls and carotenoids. In vivo, these proteins are folded with pigments to form complexes which are inserted in the thylakoid membrane of the chloroplast. The high similarity in the chemical and physical properties of the members of the family, together with the fact that they can easily lose pigments during isolation, makes their purification in a native state challenging. An alternative approach to obtain homogeneous preparations of LHCs was developed by Plumley and Schmidt in 19871, who showed that it was possible to reconstitute these complexes in vitro starting from purified pigments and unfolded apoproteins, resulting in complexes with properties very similar to that of native complexes. This opened the way to the use of bacterial expressed recombinant proteins for in vitro reconstitution. The reconstitution method is powerful for various reasons: (1) pure preparations of individual complexes can be obtained, (2) pigment composition can be controlled to assess their contribution to structure and function, (3) recombinant proteins can be mutated to study the functional role of the individual residues (e.g., pigment binding sites) or protein domain (e.g., protein-protein interaction, folding). This method has been optimized in several laboratories and applied to most of the light-harvesting complexes. The protocol described here details the method of reconstituting light-harvesting complexes in vitro currently used in our laboratory, and examples describing applications of the method are provided.

In Vitro Reconstitution of Light-harvesting Complexes of Plants and Green Algae

This protocol details the reconstitution of light-harvesting complexes in vitro. These integral membrane proteins coordinate chlorophylls and carotenoids and are responsible for harvesting light in higher plants and green algae.

식물과 녹조류의 빛 수확 단지의 체외의 재구성에

In plants and green algae, light is captured by the light-harvesting complexes (LHCs), a family of integral membrane proteins that coordinate chlorophylls ...

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.

정제 된 야생형과 돌연변이 체 CFTR 단백질의 기능의 재구성 및 채널 활동 측정

The Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) is a unique channel-forming member of the ATP Binding Cassette (ABC) superfamily of ...

Growth-based Determination and Biochemical Confirmation of Genetic Requirements for Protein Degradation in Saccharomyces cerevisiae

Regulated protein degradation is crucial for virtually every cellular function. Much of what is known about the molecular mechanisms and genetic requirements for eukaryotic protein degradation was initially established in Saccharomyces cerevisiae. Classical analyses of protein degradation have relied on biochemical pulse-chase and cycloheximide-chase methodologies. While these techniques provide sensitive means for observing protein degradation, they are laborious, time-consuming, and low-throughput. These approaches are not amenable to rapid or large-scale screening for mutations that prevent protein degradation. Here, a yeast growth-based assay for the facile identification of genetic requirements for protein degradation is described. In this assay, a reporter enzyme required for growth under specific selective conditions is fused to an unstable protein. Cells lacking the endogenous reporter enzyme but expressing the fusion protein can grow under selective conditions only when the fusion protein is stabilized (i.e. when protein degradation is compromised). In the growth assay described here, serial dilutions of wild-type and mutant yeast cells harboring a plasmid encoding a fusion protein are spotted onto selective and non-selective medium. Growth under selective conditions is consistent with degradation impairment by a given mutation. Increased protein abundance should be biochemically confirmed. A method for the rapid extraction of yeast proteins in a form suitable for electrophoresis and western blotting is also demonstrated. A growth-based readout for protein stability, combined with a simple protocol for protein extraction for biochemical analysis, facilitates rapid identification of genetic requirements for protein degradation. These techniques can be adapted to monitor degradation of a variety of short-lived proteins. In the example presented, the His3 enzyme, which is required for histidine biosynthesis, was fused to Deg1-Sec62. Deg1-Sec62 is targeted for degradation after it aberrantly engages the endoplasmic reticulum translocon. Cells harboring Deg1-Sec62-His3 were able to grow under selective conditions when the protein was stabilized.

Growth-based Determination and Biochemical Confirmation of Genetic Requirements for Protein Degradation in Saccharomyces cerevisiae

This article describes a yeast growth-based assay for the determination of genetic requirements for protein degradation. It also demonstrates a method for rapid extraction of yeast proteins, suitable for western blotting to biochemically confirm degradation requirements. These techniques can be adapted to monitor degradation of a variety of proteins.