Strategies for the Cell-Free Expression of Membrane Proteins

2009 ◽  
pp. 187-212 ◽  
Author(s):  
Sina Reckel ◽  
Solmaz Sobhanifar ◽  
Florian Durst ◽  
Frank Löhr ◽  
Vladimir A. Shirokov ◽  
...  
Keyword(s):  
Membrane Proteins ◽  
Free Expression

Cell-free expression profiling of E. coli inner membrane proteins

PROTEOMICS ◽  
2010 ◽  
Vol 10 (9) ◽  
pp. 1762-1779 ◽  
Author(s):  
Daniel Schwarz ◽  
Daniel Daley ◽  
Tobias Beckhaus ◽  
Volker Dötsch ◽  
Frank Bernhard
Keyword(s):  
Membrane Proteins ◽  
Expression Profiling ◽  
Free Expression ◽  
Inner Membrane ◽  
E Coli

scholarly journals Selective Methyl Labeling of Eukaryotic Membrane Proteins Using Cell-Free Expression

2014 ◽  
Vol 136 (32) ◽  
pp. 11308-11310 ◽  
Author(s):  
Rasmus Linser ◽  
Vladimir Gelev ◽  
Franz Hagn ◽  
Haribabu Arthanari ◽  
Sven G. Hyberts ◽  
...  
Keyword(s):  
Membrane Proteins ◽  
Free Expression ◽  
Methyl Labeling ◽  
Eukaryotic Membrane Proteins

scholarly journals Enhancing the cell-free expression of native membrane proteins by in-silico optimization of the coding sequence – an experimental study of the human voltage-dependent anion channel

2018 ◽  
Author(s):  
Sonja Zayni ◽  
Samar Damiati ◽  
Susana Moreno-Flores ◽  
Fabian Amman ◽  
Ivo Hofacker ◽  
...  
Keyword(s):  
Membrane Proteins ◽  
Translation Initiation ◽  
Scoring Function ◽  
Anion Channel ◽  
Cellular Systems ◽  
Free Expression ◽  
Voltage Dependent Anion Channel ◽  
In Vitro Synthesis ◽  
Voltage Dependent ◽  
The Impact

AbstractThe investigation of membrane proteins, key constituents of cells, is hampered by the difficulty and complexity of their in vitro synthesis, of unpredictable yield. Cell-free synthesis is herein employed to unravel the impact of the expression construct on gene transcription and translation, without the complex regulatory mechanisms of cellular systems. Through the systematic design of plasmids in the immediacy of the start of the target gene, it was possible to identify translation initiation and the conformation of mRNA as the main factors governing the cell-free expression efficiency of the human voltage dependent anion channel (VDAC), a relevant membrane protein in drug-based therapy. A simple translation initiation model was developed to quantitatively assess the expression potential for the designed constructs. A scoring function is proposed that quantifies the feasibility of formation of the translation initiation complex through the ribosome-mRNA hybridization energy and the accessibility of the mRNA segment binding to the ribosome. The scoring function enables to optimize plasmid sequences and semi-quantitatively predict protein expression efficiencies.

Cell-Free Expression of Soluble and Membrane Proteins in an Array Device for Drug Screening

2010 ◽  
Vol 82 (16) ◽  
pp. 7021-7026 ◽  
Author(s):  
Ruba Khnouf ◽  
Daniel Olivero ◽  
Shouguang Jin ◽  
Matthew A. Coleman ◽  
Z. Hugh Fan
Keyword(s):  
Membrane Proteins ◽  
Drug Screening ◽  
Free Expression

scholarly journals Direct reconstitution and study of SUN protein interactions in vitro using mammalian cell-free expression

2021 ◽  
Author(s):  
Sagardip Majumder ◽  
Yen-Yu Hsu ◽  
Allen P Liu
Keyword(s):  
Membrane Proteins ◽  
Mammalian Cell ◽  
Protein Function ◽  
Mammalian Cells ◽  
Expression System ◽  
Full Length ◽  
Free Expression ◽  
Lipid Bilayer Membranes ◽  
Linc Complex

SUN proteins are an integral part of LINC (Linker of Nucleoskeleton and Cytoskeleton) complex which spans the nuclear envelope and acts as a physical tether between the cytoskeletal filaments and the nuclear lamina. Several human diseases associated with nuclear deformation are primarily caused by impaired functioning of SUN proteins. Studies in yeast and mammalian cells have illustrated the detrimental effects of different SUN mutants in nuclear positioning and movement. While cell-based studies provide physiological relevance to the functioning of a protein, in vitro reconstitution of isolated proteins is useful in mechanistically dissecting protein function in a biochemically defined environment. In this study, we used a mammalian cell-free expression system to synthesize and reconstitute SUN proteins in artificial lipid bilayer membranes. Building on our previous work demonstrating directional reconstitution of full-length SUN proteins, we deciphered the mechanism of such protein reconstitution and leveraged it to test several theories/models of LINC complex assembly. By using a simple fluorescence-based assay, we revealed the importance of cations such as calcium and the presence of disulfide bonds in the formation of LINC complexes. Through sequential reconstitutions of SUN proteins and soluble luminal domains of SUN proteins, we found that coiled coil domains of SUN proteins are necessary for homomeric and heteromeric interactions of reconstituted SUN proteins. Overall, our results provide mechanistic insights on LINC complex formation and how this might impact cellular mechanotransduction. The facile approach for reconstituting full-length membrane proteins can be extended to study other difficult-to-study membrane proteins in vitro.

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