scholarly journals Expressing Biologically Active Membrane Proteins in a Cell-Free Transcription-Translation Platform

2017 ◽  
Author(s):  
Shaobin Guo ◽  
Amit Vaish ◽  
Qing Chen ◽  
Richard M. Murray
Keyword(s):  
Membrane Proteins ◽  
Membrane Protein ◽  
Specific Binding ◽  
Expression System ◽  
Point Of View ◽  
Biologically Active ◽  
Free Expression ◽  
Single Chain ◽  
Active Membrane ◽  
Free Environment

AbstractCell-free transcription-translation platforms have been widely utilized to express soluble proteins in basic synthetic biological circuit prototyping. From a synthetic biology point of view, it is critical to express membrane proteins in cell-free transcription-translation systems, and use them directly in biocircuits, considering the fact that histidine kinases, G-protein coupled receptors (GPCRs) and other important biosensors are all membrane proteins. Previous studies have expressed membrane proteins in cell-free systems with the help of detergents, liposomes or nanodiscs, but have not demonstrated the ability to prototype circuit behavior for the purpose of testing more complex circuit functions involving membrane-bound proteins. Built on previous efforts, in this work we demonstrated that we could co-translationally express solubilized and active membrane proteins in our cell-free TX-TL platform with membrane-like materials. We first tested the expression of several constructs with β1 and β2 adrenergic receptors in TX-TL and observed significant insoluble membrane protein production. The addition of nanodiscs to the cell free expression system enabled solubilization of membrane proteins. Nanodisc is lipoprotein-based membrane-like material. The activity of β2 adrenergic receptor was tested with both fluorescence and Surface Plasmon Resonance (SPR) binding assays by monitoring the specific binding response of small-molecule binders, carazolol and norepinephrine. Our results suggest that it is promising to use cell-free expression systems to prototype synthetic biocircuits involving single chain membrane proteins without extra procedures. This data made us one step closer to testing complex membrane protein circuits in cell-free environment.

scholarly journals Translational Fusion of Two β-Subunits of Human Chorionic Gonadotropin Results in Production of a Novel Antagonist of the Hormone

Endocrinology ◽  
2007 ◽  
Vol 148 (8) ◽  
pp. 3977-3986 ◽  
Author(s):  
Satarupa Roy ◽  
Sunita Setlur ◽  
Rupali A. Gadkari ◽  
H. N. Krishnamurthy ◽  
Rajan R. Dighe
Keyword(s):  
Chorionic Gonadotropin ◽  
Human Chorionic Gonadotropin ◽  
Expression System ◽  
Biologically Active ◽  
Chain Molecule ◽  
Dependent Manner ◽  
Β Subunit ◽  
Single Chain ◽  
Α Subunit ◽  
Lh Receptor

The strategy of translationally fusing the α- and β-subunits of human chorionic gonadotropin (hCG) into a single-chain molecule has been used to produce novel analogs of hCG. Previously we reported expression of a biologically active single-chain analog hCGαβ expressed using Pichia expression system. Using the same expression system, another analog, in which the α-subunit was replaced with the second β-subunit, was expressed (hCGββ) and purified. hCGββ could bind to LH receptor with an affinity three times lower than that of hCG but failed to elicit any response. However, it could inhibit response to the hormone in vitro in a dose-dependent manner. Furthermore, it inhibited response to hCG in vivo indicating the antagonistic nature of the analog. However, it was unable to inhibit human FSH binding or response to human FSH, indicating the specificity of the effect. Characterization of hCGαβ and hCGββ using immunological tools showed alterations in the conformation of some of the epitopes, whereas others were unaltered. Unlike hCG, hCGββ interacts with two LH receptor molecules. These studies demonstrate that the presence of the second β-subunit in the single-chain molecule generated a structure that can be recognized by the receptor. However, due to the absence of α-subunit, the molecule is unable to elicit response. The strategy of fusing two β-subunits of glycoprotein hormones can be used to produce antagonists of these hormones.

Database Study on the Expression and Purification of Membrane Proteins

2021 ◽  
Vol 28 ◽  
Author(s):  
Chen-Yan china Zhang ◽  
Shi-Qi Zhao ◽  
Shi-Long Zhang ◽  
Li-Heng Luo ◽  
Ding-Chang Liu ◽  
...  
Keyword(s):  
Membrane Proteins ◽  
Membrane Protein ◽  
Protein Expression ◽  
Drug Targets ◽  
Expression System ◽  
Data Bank ◽  
Hek293 Cells ◽  
Expression And Purification ◽  
Beta Barrel ◽  
Membrane Protein Expression

: Membrane proteins are crucial for biological processes, and many of them are important to drug targets. Understanding the three-dimensional structures of membrane proteins are essential to evaluate their bio function and drug design. High-purity membrane proteins are important for structural determination. Membrane proteins have low yields and are difficult to purify because they tend to aggregate. We summarized membrane protein expression systems, vectors, tags, and detergents, which have deposited in the Protein Data Bank (PDB) in recent four-and-a-half years. Escherichia coli is the most expression system for membrane proteins, and HEK293 cells are the most commonly cell lines for human membrane protein expression. The most frequently vectors are pFastBac1 for alpha-helical membrane proteins, pET28a for beta-barrel membrane proteins, and pTRC99a for monotopic membrane proteins. The most used tag for membrane proteins is the 6×His-tag. FLAG commonly used for alpha-helical membrane proteins, Strep and GST for beta-barrel and monotopic membrane proteins, respectively. The detergents and their concentrations used for alpha-helical, beta-barrel, and monotopic membrane proteins are different, and DDM is commonly used for membrane protein purification. It can guide the expression and purification of membrane proteins, thus contributing to their structure and bio function studying.

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.

scholarly journals Membrane protein extraction and purification using styrene–maleic acid (SMA) copolymer: effect of variations in polymer structure

2016 ◽  
Vol 473 (23) ◽  
pp. 4349-4360 ◽  
Author(s):  
Kerrie A. Morrison ◽  
Aneel Akram ◽  
Ashlyn Mathews ◽  
Zoeya A. Khan ◽  
Jaimin H. Patel ◽  
...  
Keyword(s):  
Membrane Proteins ◽  
Membrane Protein ◽  
Maleic Acid ◽  
Protein Extraction ◽  
Affinity Purification ◽  
Expression System ◽  
Transmembrane Proteins ◽  
Membrane Extraction ◽  
Total Size ◽  
Average Molecular Mass

The use of styrene–maleic acid (SMA) copolymers to extract and purify transmembrane proteins, while retaining their native bilayer environment, overcomes many of the disadvantages associated with conventional detergent-based procedures. This approach has huge potential for the future of membrane protein structural and functional studies. In this investigation, we have systematically tested a range of commercially available SMA polymers, varying in both the ratio of styrene and maleic acid and in total size, for the ability to extract, purify and stabilise transmembrane proteins. Three different membrane proteins (BmrA, LeuT and ZipA), which vary in size and shape, were used. Our results show that several polymers, can be used to extract membrane proteins, comparably to conventional detergents. A styrene:maleic acid ratio of either 2:1 or 3:1, combined with a relatively small average molecular mass (7.5–10 kDa), is optimal for membrane extraction, and this appears to be independent of the protein size, shape or expression system. A subset of polymers were taken forward for purification, functional and stability tests. Following a one-step affinity purification, SMA 2000 was found to be the best choice for yield, purity and function. However, the other polymers offer subtle differences in size and sensitivity to divalent cations that may be useful for a variety of downstream applications.

scholarly journals Expression and characterization of rat kallikrein-binding protein in Escherichia coli

1993 ◽  
Vol 292 (3) ◽  
pp. 825-832 ◽  
Author(s):  
J X Ma ◽  
L Chao ◽  
G Zhou ◽  
J Chao
Keyword(s):  
Escherichia Coli ◽  
Recombinant Protein ◽  
Specific Binding ◽  
Binding Protein ◽  
Expression System ◽  
Biologically Active ◽  
Major Protein ◽  
Tissue Kallikrein ◽  
Rat Serum ◽  
Apparent Homogeneity

Rat kallikrein-binding protein is a novel serine-proteinase inhibitor that forms a covalent complex with tissue kallikrein. We have purified rat kallikrein-binding protein and cloned the cDNA and the gene encoding rat kallikrein-binding protein [Chao, Chai, Chen, Xiong, Chao, Woodley-Miller, Wang, Lu and Chao (1990) J. Biol. Chem. 265, 16394-16401; Chai, Ma, Murray, Chao and Chao (1991) J. Biol. Chem. 266, 16029-16036]. In the present study, we have expressed rat kallikrein-binding protein in Escherichia coli with a T7-polymerase/promoter expression system. A high level of expression was detected by an e.l.i.s.a. with an average of 24.2 mg of recombinant rat kallikrein-binding protein per 1 of culture. The recombinant protein appeared as a major protein in a crude extract of Escherichia coli on SDS/PAGE. It showed a molecular mass of 43 kDa and was recognized by polyclonal antibody to the native rat kallikrein-binding protein in Western-blot analysis. The recombinant rat kallikrein-binding protein has been purified to apparent homogeneity by DEAE-Sepharose CL-6B, hydroxyapatite Bio-Gel HPHT and Mono P 5/5 column chromatography. The purified recombinant rat kallikrein-binding protein showed immunological identity with the native rat kallikrein-binding protein purified from rat serum, in a specific e.l.i.s.a. To confirm the fidelity of the expression, the N-terminal ten amino acids of the recombinant rat kallikrein-binding protein were sequenced and were shown to match perfectly with those of the native rat kallikrein-binding protein. The purified recombinant rat kallikrein-binding protein formed SDS- and heat-stable complexes with rat tissue kallikrein (rK1) and T-kininogenase (rK10) in vitro, but not with other enzymes in the rat kallikrein gene family, such as tonin (rK2) and S3 protein (rK9), which indicates enzyme-specific binding. The properties of the recombinant rat kallikrein-binding protein including its size, charge, complex formation with target enzymes and immunological characteristics were compared with those of the native protein. This expression system provides a simple way to obtain a large amount of the biologically active recombinant protein, to study structure-function relationships of the rat kallikrein-binding protein and its interaction with its target enzymes.

scholarly journals Cell-free expression of the outer membrane protein OprF of Pseudomonas aeruginosa for vaccine purposes

2021 ◽  
Vol 4 (6) ◽  
pp. e202000958
Author(s):  
Géraldine Mayeux ◽  
Landry Gayet ◽  
Lavinia Liguori ◽  
Marine Odier ◽  
Donald K Martin ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Membrane Protein ◽  
Outer Membrane ◽  
Outer Membrane Protein ◽  
Biofilm Formation ◽  
Expression System ◽  
Free Expression ◽  
Membrane Antigens ◽  
Hospital Stays ◽  
Cell Free Expression System

Pseudomonas aeruginosa is the second-leading cause of nosocomial infections and pneumonia in hospitals. Because of its extraordinary capacity for developing resistance to antibiotics, treating infections by Pseudomonas is becoming a challenge, lengthening hospital stays, and increasing medical costs and mortality. The outer membrane protein OprF is a well-conserved and immunogenic porin playing an important role in quorum sensing and in biofilm formation. Here, we used a bacterial cell-free expression system to reconstitute OprF under its native forms in liposomes and we demonstrated that the resulting OprF proteoliposomes can be used as a fully functional recombinant vaccine against P. aeruginosa. Remarkably, we showed that our system promotes the folding of OprF into its active open oligomerized state as well as the formation of mega-pores. Our approach thus represents an easy and efficient way for producing bacterial membrane antigens exposing native epitopes for vaccine purposes.

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