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1.
Methods Mol Biol ; 498: 273-96, 2009.
Article in English | MEDLINE | ID: mdl-18988032

ABSTRACT

Membrane-associated proteins and protein complexes account for approximately a third or more of the proteins in the cell (1, 2). These complexes mediate essential cellular processes; including signal transduc-tion, transport, recognition, bioenergetics and cell-cell communication. In general, membrane proteins are challenging to study because of their insolubility and tendency to aggregate when removed from their protein lipid bilayer environment. This chapter is focused on describing a novel method for producing and solubilizing membrane proteins that can be easily adapted to high-throughput expression screening. This process is based on cell-free transcription and translation technology coupled with nanolipoprotein par ticles (NLPs), which are lipid bilayers confined within a ring of amphipathic protein of defined diameter. The NLPs act as a platform for inserting, solubilizing and characterizing functional membrane proteins. NLP component proteins (apolipoproteins), as well as membrane proteins can be produced by either traditional cell-based or as discussed here, cell-free expression methodologies.


Subject(s)
Lipoproteins/metabolism , Membrane Proteins/isolation & purification , Recombinant Proteins/isolation & purification , Animals , Biotinylation , Cell Fractionation/methods , Escherichia coli/genetics , Lipoproteins/chemistry , Membrane Proteins/biosynthesis , Membrane Proteins/metabolism , Nanoparticles/chemistry , Protein Array Analysis , Recombinant Proteins/biosynthesis , Recombinant Proteins/metabolism , Solubility
3.
Mol Cell Proteomics ; 7(11): 2246-53, 2008 Nov.
Article in English | MEDLINE | ID: mdl-18603642

ABSTRACT

Here we demonstrate rapid production of solubilized and functional membrane protein by simultaneous cell-free expression of an apolipoprotein and a membrane protein in the presence of lipids, leading to the self-assembly of membrane protein-containing nanolipoprotein particles (NLPs). NLPs have shown great promise as a biotechnology platform for solubilizing and characterizing membrane proteins. However, current approaches are limited because they require extensive efforts to express, purify, and solubilize the membrane protein prior to insertion into NLPs. By the simple addition of a few constituents to cell-free extracts, we can produce membrane proteins in NLPs with considerably less effort. For this approach an integral membrane protein and an apolipoprotein scaffold are encoded by two DNA plasmids introduced into cell-free extracts along with lipids. For this study reported here we used plasmids encoding the bacteriorhodopsin (bR) membrane apoprotein and scaffold protein Delta1-49 apolipoprotein A-I fragment (Delta49A1). Cell free co-expression of the proteins encoded by these plasmids, in the presence of the cofactor all-trans-retinal and dimyristoylphosphatidylcholine, resulted in production of functional bR as demonstrated by a 5-nm shift in the absorption spectra upon light adaptation and characteristic time-resolved FT infrared difference spectra for the bR --> M transition. Importantly the functional bR was solubilized in discoidal bR.NLPs as determined by atomic force microscopy. A survey study of other membrane proteins co-expressed with Delta49A1 scaffold protein also showed significantly increased solubility of all of the membrane proteins, indicating that this approach may provide a general method for expressing membrane proteins enabling further studies.


Subject(s)
Apolipoprotein A-I/chemistry , Membrane Proteins/chemistry , Apolipoprotein A-I/genetics , Bacteriorhodopsins/chemistry , Bacteriorhodopsins/genetics , Base Sequence , DNA Primers/genetics , Halobacterium salinarum/genetics , Membrane Proteins/genetics , Microscopy, Atomic Force , Nanoparticles/chemistry , Peptide Fragments/chemistry , Peptide Fragments/genetics , Proteomics , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Solubility , Spectroscopy, Fourier Transform Infrared
4.
J Proteome Res ; 7(8): 3535-42, 2008 Aug.
Article in English | MEDLINE | ID: mdl-18557639

ABSTRACT

We report a cell-free approach for expressing and inserting integral membrane proteins into water-soluble particles composed of discoidal apolipoprotein-lipid bilayers. Proteins are inserted into the particles, circumventing the need of extracting and reconstituting the product into membrane vesicles. Moreover, the planar nature of the membrane support makes the protein freely accessible from both sides of the lipid bilayer. Complexes are successfully purified by means of the apoplipoprotein component or by the carrier protein. The method significantly enhances the solubility of a variety of membrane proteins with different functional roles and topologies. Analytical assays for a subset of model membrane proteins indicate that proteins are correctly folded and active. The approach provides a platform amenable to high-throughput structural and functional characterization of a variety of traditionally intractable drug targets.


Subject(s)
Lipid Bilayers/chemistry , Membrane Proteins/chemistry , Antiporters/biosynthesis , Antiporters/chemistry , Antiporters/genetics , Apolipoprotein A-I/biosynthesis , Apolipoprotein A-I/chemistry , Apolipoprotein A-I/genetics , Apolipoprotein E4/biosynthesis , Apolipoprotein E4/chemistry , Apolipoprotein E4/genetics , Bacteriorhodopsins/biosynthesis , Bacteriorhodopsins/chemistry , Bacteriorhodopsins/genetics , Chromatography, Gel , Escherichia coli Proteins/biosynthesis , Escherichia coli Proteins/chemistry , Escherichia coli Proteins/genetics , Humans , Membrane Proteins/biosynthesis , Membrane Proteins/genetics , Microscopy, Atomic Force , Solubility
5.
J Biol Chem ; 279(36): 37852-9, 2004 Sep 03.
Article in English | MEDLINE | ID: mdl-15229221

ABSTRACT

Five components have thus far been identified that are necessary for the incorporation of selenocysteine (Sec) into approximately 25 mammalian proteins. Two of these are cis sequences, a SECIS element in the 3'-untranslated region and a Sec codon (UGA) in the coding region. The three known trans-acting factors are a Sec-specific translation elongation factor (eEFSec), the Sec-tRNA(Sec), and a SECIS-binding protein, SBP2. Here we describe a system in which the efficiency of Sec incorporation was determined quantitatively both in vitro and in transfected cells, and in which the contribution of each of the known factors is examined. The efficiency of Sec incorporation into a luciferase reporter system in vitro is maximally 5-8%, which is 6-10 times higher than that in transfected rat hepatoma cells, McArdle 7777. In contrast, the efficiency of Sec incorporation into selenoprotein P in vitro is approximately 40%, suggesting that as yet unidentified cis-elements may regulate differential selenoprotein expression. In addition, we have found that SBP2 is the only limiting factor in rabbit reticulocyte lysate but not in transfected rat hepatoma cells where SBP2 is found to be mostly if not entirely cytoplasmic despite having a strong putative nuclear localization signal. The significance of these findings with regard to the function of known Sec incorporation factors is discussed.


Subject(s)
Selenocysteine/metabolism , Animals , Cell Line, Tumor , Plasmids , RNA, Messenger/genetics , Rats , Subcellular Fractions/metabolism
6.
J Immunol ; 168(5): 2456-63, 2002 Mar 01.
Article in English | MEDLINE | ID: mdl-11859138

ABSTRACT

Although well recognized for its anti-inflammatory effect on gene expression in stimulated monocytes and macrophages, IL-4 is a pleiotropic cytokine that has also been shown to enhance TNF-alpha and IL-12 production in response to stimulation with LPS. In the present study we expand these prior studies in three areas. First, the potentiating effect of IL-4 pretreatment is both stimulus and gene selective. Pretreatment of mouse macrophages with IL-4 for a minimum of 6 h produces a 2- to 4-fold enhancement of LPS-induced expression of several cytokines and chemokines, including TNF-alpha, IL-1alpha, macrophage-inflammatory protein-2, and KC, but inhibits the production of IL-12p40. In addition, the production of TNF-alpha by macrophages stimulated with IFN-gamma and IL-2 is inhibited by IL-4 pretreatment, while responses to both LPS and dsRNA are enhanced. Second, the ability of IL-4 to potentiate LPS-stimulated cytokine production appears to require new IL-4-stimulated gene expression, because it is time dependent, requires the activation of STAT6, and is blocked by the reversible protein synthesis inhibitor cycloheximide during the IL-4 pretreatment period. Finally, IL-4-mediated potentiation of TNF-alpha production involves specific enhancement of mRNA translation. Although TNF-alpha protein is increased in IL-4-pretreated cells, the level of mRNA remains unchanged. Furthermore, LPS-stimulated TNF-alpha mRNA is selectively enriched in actively translating large polyribosomes in IL-4-pretreated cells compared with cells stimulated with LPS alone.


Subject(s)
Chemokines/biosynthesis , Cytokines/biosynthesis , Interleukin-4/pharmacology , Lipopolysaccharides/pharmacology , Macrophages, Peritoneal/immunology , Animals , Cells, Cultured , Chemokines/genetics , Cytokines/genetics , Drug Synergism , Kinetics , Macrophages, Peritoneal/drug effects , Mice , Mice, Inbred C57BL , Mice, Knockout , Protein Biosynthesis/drug effects , RNA, Messenger/biosynthesis , STAT6 Transcription Factor , Trans-Activators/genetics , Trans-Activators/physiology , Transcriptional Activation , Tumor Necrosis Factor-alpha/biosynthesis , Tumor Necrosis Factor-alpha/genetics
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