scholarly journals The use of blue native PAGE in the evaluation of membrane protein aggregation states for crystallization

2008 ◽  
Vol 41 (6) ◽  
pp. 1150-1160 ◽  
Author(s):  
Jichun Ma ◽  
Di Xia
Keyword(s):  
Membrane Proteins ◽  
Membrane Protein ◽  
Protein Quality ◽  
Structural Information ◽  
Protein Complexes ◽  
Polyacrylamide Gel Electrophoresis ◽  
Size Exclusion ◽  
Native Page ◽  
Exclusion Chromatography ◽  
Blue Native

Crystallization has long been one of the bottlenecks in obtaining structural information at atomic resolution for membrane proteins. This is largely due to difficulties in obtaining high-quality protein samples. One frequently used indicator of protein quality for successful crystallization is the monodispersity of proteins in solution, which is conventionally obtained by size exclusion chromatography (SEC) or by dynamic light scattering (DLS). Although useful in evaluating the quality of soluble proteins, these methods are not always applicable to membrane proteins either because of the interference from detergent micelles or because of the requirement for large sample quantities. Here, the use of blue native polyacrylamide gel electrophoresis (BN–PAGE) to assess aggregation states of membrane protein samples is reported. A strong correlation is demonstrated between the monodispersity measured by BN–PAGE and the propensity for crystallization of a number of soluble and membrane protein complexes. Moreover, it is shown that there is a direct correspondence between the oligomeric states of proteins as measured by BN–PAGE and those obtained from their crystalline forms. When applied to a membrane protein with unknown structure, BN–PAGE was found to be useful and efficient for selecting well behaved proteins from various constructs and in screening detergents. Comparisons of BN–PAGE with DLS and SEC are provided.

scholarly journals Fluorescence-detection size-exclusion chromatography utilizing nanobody technology for expression screening of membrane proteins

2020 ◽  
Author(s):  
Fei Jin ◽  
Yao Wang ◽  
Mengqi Wang ◽  
Minxuan Sun ◽  
Motoyuki Hattori
Keyword(s):  
Membrane Proteins ◽  
Membrane Protein ◽  
Protein Expression ◽  
Size Exclusion Chromatography ◽  
Fluorescence Detection ◽  
Size Exclusion ◽  
Expression Screening ◽  
Functional Studies ◽  
Membrane Protein Expression ◽  
Exclusion Chromatography

AbstractMembrane proteins play numerous physiological roles and are thus of tremendous interest in pharmacology. Nevertheless, stable and homogeneous sample preparation is one of the bottlenecks in biophysical and pharmacological studies of membrane proteins because membrane proteins are typically unstable and poorly expressed. To overcome such obstacles, GFP fusion-based Fluorescence-detection Size-Exclusion Chromatography (FSEC) has been widely employed for membrane protein expression screening for over a decade. However, fused GFP itself may occasionally affect the expression and/or stability of the targeted membrane protein, leading to both false-positive and false-negative results in expression screening. Furthermore, GFP fusion technology is not well suited for some membrane proteins depending on their membrane topology. Here, we developed an FSEC assay utilizing nanobody (Nb) technology, named FSEC-Nb, in which targeted membrane proteins are fused to a small peptide tag and recombinantly expressed. The whole-cell extracts are solubilized, mixed with anti-peptide Nb fused to GFP and applied to a size-exclusion chromatography column attached to a fluorescence detector for FSEC analysis. FSEC-Nb enables one to evaluate the expression, monodispersity and thermostability of membrane proteins without the need of purification by utilizing the benefits of the GFP fusion-based FSEC method, but does not require direct GFP fusion to targeted proteins. We applied FSEC-Nb to screen zinc-activated ion channel (ZAC) family proteins in the Cys-loop superfamily and membrane proteins from SARS-CoV-2 as examples of the practical application of FSEC-Nb. We successfully identified a ZAC ortholog with high monodispersity but moderate expression levels that could not be identified with the previously developed GFP fusion-free FSEC method. Consistent with the results of FSEC-Nb screening, the purified ZAC ortholog showed monodispersed particles by both negative staining EM and cryo-EM. Furthermore, we identified two membrane proteins from SARS-CoV-2 with high monodispersity and expression level by FSEC-Nb, which may facilitate structural and functional studies of SARS-CoV-2. Overall, our results show FSEC-Nb as a powerful tool for membrane protein expression screening that can provide further opportunity to prepare well-behaved membrane proteins for structural and functional studies.

Analysis of Membrane Protein Complexes by Blue Native PAGE

PROTEOMICS ◽  
2006 ◽  
Vol 6 (S2) ◽  
pp. 6-15 ◽  
Author(s):  
Veronika Reisinger ◽  
Lutz Andreas Eichacker
Keyword(s):  
Membrane Protein ◽  
Protein Complexes ◽  
Native Page ◽  
Blue Native Page ◽  
Membrane Protein Complexes ◽  
Blue Native

Analysis of membrane-protein complexes of the marine sulfate reducer Desulfobacula toluolica Tol2 by 1D blue native-PAGE complexome profiling and 2D blue native-/SDS-PAGE

PROTEOMICS ◽  
2016 ◽  
Vol 16 (6) ◽  
pp. 973-988 ◽  
Author(s):  
Lars Wöhlbrand ◽  
Hanna S. Ruppersberg ◽  
Christoph Feenders ◽  
Bernd Blasius ◽  
Hans-Peter Braun ◽  
...  
Keyword(s):  
Membrane Protein ◽  
Protein Complexes ◽  
Sulfate Reducer ◽  
Native Page ◽  
Blue Native Page ◽  
Sds Page ◽  
Membrane Protein Complexes ◽  
Blue Native ◽  
Desulfobacula Toluolica

Blue native-PAGE analysis of membrane protein complexes in Porphyromonas gingivalis

2014 ◽  
Vol 110 ◽  
pp. 72-92 ◽  
Author(s):  
Michelle D. Glew ◽  
Paul D. Veith ◽  
Dina Chen ◽  
Christine A. Seers ◽  
Yu-Yen Chen ◽  
...  
Keyword(s):  
Membrane Protein ◽  
Porphyromonas Gingivalis ◽  
Protein Complexes ◽  
Native Page ◽  
Blue Native Page ◽  
Membrane Protein Complexes ◽  
Blue Native

scholarly journals Fluorescence-detection size-exclusion chromatography utilizing nanobody technology for expression screening of membrane proteins

2021 ◽  
Vol 4 (1) ◽  
Author(s):  
Fei Jin ◽  
Cheng Shen ◽  
Yao Wang ◽  
Mengqi Wang ◽  
Minxuan Sun ◽  
...  
Keyword(s):  
Membrane Proteins ◽  
Membrane Protein ◽  
Size Exclusion Chromatography ◽  
Fluorescence Detection ◽  
False Negative ◽  
Membrane Topology ◽  
Size Exclusion ◽  
Expression Screening ◽  
Cell Extracts ◽  
Exclusion Chromatography

AbstractGFP fusion-based fluorescence-detection size-exclusion chromatography (FSEC) has been widely employed for membrane protein expression screening. However, fused GFP itself may occasionally affect the expression and/or stability of the targeted membrane protein, leading to both false-positive and false-negative results in expression screening. Furthermore, GFP fusion technology is not well suited for some membrane proteins, depending on their membrane topology. Here, we developed an FSEC assay utilizing nanobody (Nb) technology, named FSEC-Nb, in which targeted membrane proteins are fused to a small peptide tag and recombinantly expressed. The whole-cell extracts are solubilized, mixed with anti-peptide Nb fused to GFP for FSEC analysis. FSEC-Nb enables the evaluation of the expression, monodispersity and thermostability of membrane proteins without the need for purification but does not require direct GFP fusion to targeted proteins. Our results show FSEC-Nb as a powerful tool for expression screening of membrane proteins for structural and functional studies.

scholarly journals Studies of recombinant TWA1 reveal constitutive dimerization

2017 ◽  
Vol 37 (1) ◽  
Author(s):  
Ore Francis ◽  
Genevieve E. Baker ◽  
Paul R. Race ◽  
Josephine C. Adams
Keyword(s):  
Protein Complexes ◽  
Carbon Sources ◽  
Gel Filtration ◽  
Cd Spectroscopy ◽  
Cysteine Residue ◽  
Size Exclusion ◽  
Native Page ◽  
Exclusion Chromatography ◽  
Key Enzyme

The mammalian muskelin/RanBP9/C-terminal to LisH (CTLH) complex and the Saccharomyces cerevisiae glucose-induced degradation (GID) complex are large, multi-protein complexes that each contain a RING E3 ubiquitin ligase. The yeast GID complex acts to degrade a key enzyme of gluconeogenesis, fructose 1,6-bisphosphatase, under conditions of abundant fermentable carbon sources. However, the assembly and functions of the mammalian complex remain poorly understood. A striking feature of these complexes is the presence of multiple proteins that contain contiguous lissencephaly-1 homology (LisH), CTLH and C-terminal CT11-RanBP9 (CRA) domains. TWA1/Gid8, the smallest constituent protein of these complexes, consists only of LisH, CTLH and CRA domains and is highly conserved in eukaryotes. Towards better knowledge of the role of TWA1 in these multi-protein complexes, we established a method for bacterial expression and purification of mouse TWA1 that yields tag-free, recombinant TWA1 in quantities suitable for biophysical and biochemical studies. CD spectroscopy of recombinant TWA1 indicated a predominantly α-helical protein. Gel filtration chromatography, size-exclusion chromatography (SEC) with multi-angle light scattering (SEC-MALS) and native PAGE demonstrated a propensity of untagged TWA1 to form stable dimers and, to a lesser extent, higher order oligomers. TWA1 has a single cysteine residue, Cys139, yet the dimeric form was preserved when TWA1 was purified in the presence of the reducing agent tris(2-carboxyethyl)phosphine (TCEP). These findings have implications for understanding the molecular role of TWA1 in the yeast GID complex and related multi-protein E3 ubiquitin ligases identified in other eukaryotes.

scholarly journals A Blue Native-PAGE analysis of membrane protein complexes in Clostridium thermocellum

2011 ◽  
Vol 11 (1) ◽  
pp. 22 ◽  
Author(s):  
Yanfeng Peng ◽  
Yuanming Luo ◽  
Tingting Yu ◽  
Xinping Xu ◽  
Keqiang Fan ◽  
...  
Keyword(s):  
Membrane Protein ◽  
Clostridium Thermocellum ◽  
Protein Complexes ◽  
Native Page ◽  
Blue Native Page ◽  
Membrane Protein Complexes ◽  
Blue Native

scholarly journals Biochemical Reduction of the Topology of the Diverse WDR76 Protein Interactome

2019 ◽  
Author(s):  
Gerald Dayebgadoh ◽  
Mihaela E. Sardiu ◽  
Laurence Florens ◽  
Michael P. Washburn
Keyword(s):  
Cell Cycle Progression ◽  
Protein Quality ◽  
Gene Expression Regulation ◽  
Protein Complexes ◽  
Affinity Purification ◽  
Interaction Network ◽  
Size Exclusion ◽  
Wd40 Repeat ◽  
Exclusion Chromatography ◽  
Apoptosis Gene

AbstractA hub protein in protein interaction networks will typically have a large number of diverse interactions. Determining the core interactions and the function of such a hub protein remains a significant challenge in the study of networks. Proteins with WD40 repeats represent a large class of proteins that can be hub proteins. WDR76 is a poorly characterized WD40 repeat protein with possible involvement in DNA damage repair, cell cycle progression, apoptosis, gene expression regulation, and protein quality control. WDR76 has a large and diverse interaction network that has made its study challenging. Here, we rigorously carry out a series of affinity-purification coupled to mass spectrometry (AP-MS) to map out the WDR76 interactome through different biochemical conditions. We apply AP-MS analysis coupled to size exclusion chromatography to resolve WDR76-based protein complexes. Furthermore, we also show that WDR76 interacts with the CCT complex via its WD40 repeat domain and with DNA-PK-KU, PARP1, GAN, SIRT1, and histones outside of the WD40 domain. An evaluation of the stability of WDR76 interactions led to focused and streamlined reciprocal analyses that validate the interactions with GAN and SIRT1. Overall, the approaches used to study WDR76 would be valuable to study other proteins containing WD40 repeat domains, which are conserved in a large number of proteins in many organisms

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