Clean Western Blots of Membrane Proteins after Yeast Heterologous Expression Following a Shortened Version of the Method of Perini et al.

2000 ◽  
Vol 285 (2) ◽  
pp. 276-278 ◽  
Author(s):  
José M. Fuentes ◽  
Anne-Marie Lompré ◽  
Jesper V. Moller ◽  
Pierre Falson ◽  
Marc le Maire
Keyword(s):  
Membrane Proteins ◽  
Heterologous Expression ◽  
Western Blots

scholarly journals Temperature Regulation of the Hemin Storage (Hms+) Phenotype of Yersinia pestis Is Posttranscriptional

2004 ◽  
Vol 186 (6) ◽  
pp. 1638-1647 ◽  
Author(s):  
Robert D. Perry ◽  
Alexander G. Bobrov ◽  
Olga Kirillina ◽  
Heather A. Jones ◽  
Lisa Pedersen ◽  
...  
Keyword(s):  
Membrane Proteins ◽  
Yersinia Pestis ◽  
Growth Temperature ◽  
Temperature Regulation ◽  
Iron Status ◽  
Outer Membrane Proteins ◽  
Western Blots ◽  
Protein Levels ◽  
The Stability ◽  
In Cells

ABSTRACT In Yersinia pestis, the Congo red (and hemin) binding that is characteristic of the Hms+ phenotype occurs at temperatures up to 34°C but not at higher temperatures. Manifestation of the Hms+ phenotype requires at least five proteins (HmsH, -F, -R, -S, and -T) that are organized into two separate operons: hmsHFRS and hmsT. HmsH and HmsF are outer membrane proteins, while HmsR, HmsS, and HmsT are predicted to be inner membrane proteins. We have used transcriptional reporter constructs, RNA dot blots, and Western blots to examine the expression of hms operons and proteins. Our studies indicate that transcription from the hmsHFRS and hmsT promoters is not regulated by the iron status of the cells, growth temperature, or any of the Hms proteins. In addition, the level of mRNA for both operons is not significantly affected by growth temperature. However, protein levels of HmsH, HmsR, and HmsT in cells grown at 37°C are very low compared to those in cells grown at 26°C, while the amounts of HmsF and HmsS show only a moderate reduction at the higher growth temperature. Neither the Pla protease nor a putative endopeptidase (Y2360) encoded upstream of hmsH is essential for temperature regulation of the Hms+ phenotype. However, HmsT at 37°C is sensitive to degradation by Lon and/or ClpPX. Thus, the stability of HmsH, HmsR, and HmsT proteins likely plays a role in temperature regulation of the Hms+ phenotype of Y. pestis.

scholarly journals An Approach to Heterologous Expression of Membrane Proteins. The Case of Bacteriorhodopsin

PLoS ONE ◽  
2015 ◽  
Vol 10 (6) ◽  
pp. e0128390 ◽  
Author(s):  
Dmitry Bratanov ◽  
Taras Balandin ◽  
Ekaterina Round ◽  
Vitaly Shevchenko ◽  
Ivan Gushchin ◽  
...  
Keyword(s):  
Membrane Proteins ◽  
Heterologous Expression

scholarly journals Heterologous Expression and Purification Systems for Structural Proteomics of Mammalian Membrane Proteins

2002 ◽  
Vol 3 (6) ◽  
pp. 511-517 ◽  
Author(s):  
Isabelle Mus-Veteau
Keyword(s):  
Membrane Proteins ◽  
Heterologous Expression ◽  
Large Scale ◽  
Structural Information ◽  
Structural Data ◽  
Structural Proteomics ◽  
Scale Production ◽  
Expression And Purification ◽  
Large Scale Production ◽  
Heterologous Expression Systems

Membrane proteins (MPs) are responsible for the interface between the exterior and the interior of the cell. These proteins are implicated in numerous diseases, such as cancer, cystic fibrosis, epilepsy, hyperinsulinism, heart failure, hypertension and Alzheimer's disease. However, studies on these disorders are hampered by a lack of structural information about the proteins involved. Structural analysis requires large quantities of pure and active proteins. The majority of medically and pharmaceutically relevant MPs are present in tissues at very low concentration, which makes heterologous expression in large-scale production-adapted cells a prerequisite for structural studies. Obtaining mammalian MP structural data depends on the development of methods that allow the production of large quantities of MPs. This review focuses on the different heterologous expression systems, and the purification strategies, used to produce large amounts of pure mammalian MPs for structural proteomics.

scholarly journals Selective Enrichment of Membrane Proteins by Partition Phase Separation for Proteomic Studies

2003 ◽  
Vol 2003 (4) ◽  
pp. 249-255 ◽  
Author(s):  
M. Walid Qoronfleh ◽  
Betsy Benton ◽  
Ray Ignacio ◽  
Barbara Kaboord
Keyword(s):  
Membrane Proteins ◽  
Membrane Protein ◽  
Protein Extraction ◽  
Yeast Cells ◽  
Protein Markers ◽  
Two Phase ◽  
Western Blots ◽  
Selective Enrichment ◽  
Phase Partitioning ◽  
Hydrophobic Fraction

The human proteome project will demand faster, easier, and more reliable methods to isolate and purify protein targets. Membrane proteins are the most valuable group of proteins since they are the target for 70–80% of all drugs. Perbio Science has developed a protocol for the quick, easy, and reproducible isolation of integral membrane proteins from eukaryotic cells. This procedure utilizes a proprietary formulation to facilitate cell membrane disruption in a mild, nondenaturing environment and efficiently solubilizes membrane proteins. The technique utilizes a two-phase partitioning system that enables the class separation of hydrophobic and hydrophilic proteins. A variety of protein markers were used to investigate the partitioning efficiency of the membrane protein extraction reagents (Mem-PER) (Mem-PER is a registered trademark of Pierce Biotechnology, Inc) system. These included membrane proteins with one or more transmembrane spanning domains as well as peripheral and cytosolic proteins. Based on densitometry analyses of our Western blots, we obtained excellent solubilization of membrane proteins with less than 10% contamination of the hydrophobic fraction with hydrophilic proteins. Compared to other methodologies for membrane protein solubilization that use time-consuming protocols or expensive and cumbersome instrumentation, the Mem-PER reagents system for eukaryotic membrane protein extraction offers an easy, efficient, and reproducible method to isolate membrane proteins from mammalian and yeast cells.

scholarly journals Cloning, sequencing and heterologous expression of a cDNA encoding pigeon liver carnitine acetyltransferase

1995 ◽  
Vol 305 (2) ◽  
pp. 439-444 ◽  
Author(s):  
T M Johnson ◽  
H P Kocher ◽  
R C Anderson ◽  
G M Nemecek
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Heterologous Expression ◽  
Stop Codon ◽  
Expression System ◽  
Breast Muscle ◽  
Cdna Clones ◽  
Carnitine Acetyltransferase ◽  
Western Blots ◽  
Pigeon Liver

Two overlapping cDNA clones encoding pigeon liver carnitine acetyltransferase (EC 2.3.1.7) (CAT) were isolated from a pigeon liver lambda gt11 cDNA library by gene amplification using oligonucleotide primers based on the N-terminal amino acid sequence of the enzyme. The two clones, which represent the 5′ and 3′ ends of the gene, were spliced together to form a single cDNA construct containing the entire coding sequence for CAT, with an in-frame TGA stop codon 42 bases before the first ATG start site and a 3′-untranslated segment of 1057 bases. The largest open reading frame of 1942 nucleotides predicted a polypeptide of 627 amino acids and a molecular mass of 71.1 kDa. The N-terminus and four internal peptides from the amino acid sequence of pigeon breast muscle CAT were identified in the predicted sequence of the liver cDNA clone. The identity of the CAT cDNA was confirmed by heterologous expression of active recombinant CAT (rCAT) in insect cells using the baculovirus expression system. Western blots of rCAT from infected insect cell lysates and immunodetection with a rabbit anti-CAT polyclonal serum showed an immunoreactive protein band similar in size to native CAT from pigeon breast muscle. Like the native enzyme, rCAT was capable of acylating carnitine with a preference for small-chain acyl-CoAs of carbon chain lengths C2-C4.

scholarly journals Perturbation of the yeast mitochondrial lipidome and associated membrane proteins following heterologous expression of Artemia-ANT

2018 ◽  
Vol 8 (1) ◽  
Author(s):  
Emily Chen ◽  
Michael A. Kiebish ◽  
Justice McDaniel ◽  
Katarzyna Niedzwiecka ◽  
Roza Kucharczyk ◽  
...  
Keyword(s):  
Membrane Proteins ◽  
Heterologous Expression

Quantitation and topography of membrane proteins in highly water-permeable vesicles from ADH-stimulated toad bladder

1991 ◽  
Vol 261 (1) ◽  
pp. C143-C153 ◽  
Author(s):  
H. W. Harris ◽  
M. L. Zeidel ◽  
C. Hosselet
Keyword(s):  
Membrane Proteins ◽  
Membrane Protein ◽  
Lipid Bilayer ◽  
Apical Membrane ◽  
Water Channel ◽  
Protein S ◽  
Water Channels ◽  
Toad Bladder ◽  
Western Blots ◽  
Vesicle Protein

Antidiuretic hormone (ADH) stimulation of toad bladder granular cells rapidly increases the osmotic water permeability (Pf) of their apical membranes by insertion of highly selective water channels. Before ADH stimulation, these water channels are stored in large cytoplasmic vesicles called aggrephores. ADH causes aggrephores to fuse with the apical membrane. Termination of ADH stimulation results in prompt endocytosis of water channel-containing membranes via retrieval of these specialized regions of apical membrane. Protein components of the ADH water channel contained within these retrieved vesicles would be expected to be integral membrane protein(s) that span the vesicle's lipid bilayer to create narrow aqueous channels. Our previous work has identified proteins of 55 (actually a 55/53-kDa doublet), 17, 15, and 7 kDa as candidate ADH water channel components. We now have investigated these candidate ADH water channel proteins in purified retrieved vesicles. These vesicles do not contain a functional proton pump as assayed by Western blots of purified vesicle protein probed with anti-H(+)-ATPase antisera. Approximately 60% of vesicle protein is accounted for by three protein bands of 55, 53, and 46 kDa. Smaller contributions to vesicle protein are made by the 17- and 15-kDa proteins. Triton X-114-partitioning analysis shows that the 55, 53, 46, and 17 kDa are integral membrane proteins. Vectorial labeling analysis with two membrane-impermeant reagents shows that the 55-, 53-, and 46-kDa protein species span the lipid bilayer of these vesicles. Thus the 55-, 53-, and 46-kDa proteins possess characteristics expected for ADH water channel components. These data show that the 55- and 53- and perhaps the 46-, 17-, and 15-kDa proteins are likely components of aqueous transmembrane pores that constitute ADH water channels contained within these vesicles.

scholarly journals Characterization of a monoclonal antibody that induces the acrosome reaction of sea urchin sperm.

1987 ◽  
Vol 105 (3) ◽  
pp. 1121-1128 ◽  
Author(s):  
J S Trimmer ◽  
Y Ebina ◽  
R W Schackmann ◽  
C G Meinhof ◽  
V D Vacquier
Keyword(s):  
Monoclonal Antibody ◽  
Membrane Proteins ◽  
Sea Urchin ◽  
Acrosome Reaction ◽  
Periodate Oxidation ◽  
Western Blots ◽  
Protease Digestion ◽  
Molecular Masses ◽  
Egg Jelly ◽  
Polypeptide Chains

A monoclonal antibody, J18/29, induces the acrosome reaction (AR) in spermatozoa of the sea urchin Strongylocentrotus purpuratus. J18/29 induces increases in both intracellular Ca2+ and intracellular pH similar to those occurring upon induction of the AR by the natural inducer, the fucose sulfate-rich glycoconjugate of egg jelly. Lowering the Ca2+ concentration or the pH of the seawater inhibits the J18/29-induced AR, as does treatment with Co2+, an inhibitor of Ca2+ channels. The J18/29-induced AR is also inhibited by verapamil, tetraethylammonium chloride, and elevated K+. All these treatments cause similar inhibition of the egg jelly-induced AR. J18/29 reacts with a group of membrane proteins ranging in molecular mass from 340 to 25 kD, as shown by immunoprecipitation of lysates of 125I-labeled sperm and Western blots. The most prominent reacting proteins are of molecular masses of 320, 240, 170, and 58 kD. The basis of the multiple reactivity appears to reside in the polypeptide chains of these proteins, as J18/29 binding is sensitive to protease digestion but resistant to periodate oxidation. There are approximately 570,000 sites per cell for J18/29 binding. J18/29 is the only reagent of known binding specificity that induces the AR; it identifies a subset of sperm membrane proteins whose individual characterization may lead to the isolation of the receptors involved in the triggering of the AR at fertilization.

scholarly journals Method for Recovery and Immunoaffinity Enrichment of Membrane Proteins Illustrated with Metastatic Ovarian Cancer Tissues

2012 ◽  
Vol 2012 ◽  
pp. 1-15 ◽  
Author(s):  
Luke V. Schneider ◽  
Varsha Likhte ◽  
William H. Wright ◽  
Frances Chu ◽  
Emma Cambron ◽  
...  
Keyword(s):  
Membrane Proteins ◽  
Membrane Protein ◽  
Integral Membrane Proteins ◽  
Buffer System ◽  
Western Blots ◽  
Pathogen Invasion ◽  
Quantitative Extraction ◽  
Extraction Buffer ◽  
Pressure Cycling Technology ◽  
Cancer Tissues

Integral membrane proteins play key biological roles in cell signaling, transport, and pathogen invasion. However, quantitative clinical assays for this critical class of proteins remain elusive and are generally limited to serum-soluble extracellular fragments. Furthermore, classic proteomic approaches to membrane protein analysis typically involve proteolytic digestion of the soluble pieces, resulting in separation of intra- and extracellular segments and significant informational loss. In this paper, we describe the development of a new method for the quantitative extraction of intact integral membrane proteins (including GPCRs) from solid metastatic ovarian tumors using pressure cycling technology in combination with a new (ProteoSolve-TD) buffer system. This new extraction buffer is compatible with immunoaffinity methods (e.g., ELISA and immunoaffinity chromatography), as well as conventional proteomic techniques (e.g., 2D gels, western blots). We demonstrate near quantitative recovery of membrane proteins EDG2, EDG4, FASLG, KDR, and LAMP-3 by western blots. We have also adapted commercial ELISAs for serum-soluble membrane protein fragments (e.g., sVEGFR2) to measure the tissue titers of their transmembrane progenitors. Finally, we demonstrate the compatibility of the new buffers with immunoaffinity enrichment/mass spectrometric characterization of tissue proteins.

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