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.

Synthesis and characterization of a functional intact IgG in a prokaryotic cell-free expression system

2008 ◽  
Vol 389 (1) ◽  
pp. 37-45 ◽  
Author(s):  
Stephan Frey ◽  
Martin Haslbeck ◽  
Otmar Hainzl ◽  
Johannes Buchner
Keyword(s):  
Mammalian Cells ◽  
Recombinant Expression ◽  
Expression System ◽  
Basic Research ◽  
Medical Diagnostics ◽  
Free Expression ◽  
Translation System ◽  
Functional Antibodies ◽  
The Stability

Abstract Antibodies are an important component of the immune system of higher eukaryotes. Furthermore, they are effective tools in basic research, medical diagnostics and therapy. Recombinant expression of these heterotetrameric, disulfide-bridged proteins is usually performed in mammalian cells. Here, we describe the cell-free expression of a mouse monoclonal antibody, MAK33, in a coupled transcription/translation system, based on an Escherichia coli lysate. Both the heavy and the light chain can be produced efficiently in this setup. However, they fail to form functional antibodies. With a view to overcome folding and oxidation defects, we supplemented the system with the oxidoreductases PDI (protein disulfide isomerase) and DsbC and the ER-specific chaperones Grp94 and BiP; furthermore, we optimized the redox conditions. We found that functional antibodies can only be obtained in the presence of an oxidoreductase. In contrast, the addition of Grp94 and/or BiP had no influence on the productive folding reaction. The comparison of the antibody expressed in vitro with MAK33 expressed in cell culture showed that the in vitro expressed antibody is correctly assembled, disulfide-bridged and shows identical antigen affinity. The stability of the in vitro expressed non-glycosylated IgG is comparable to that of the authentic antibody.

scholarly journals Efficacy of a Potential Trivalent Vaccine Based on Hc Fragments of Botulinum Toxins A, B, and E Produced in a Cell-Free Expression System

2010 ◽  
Vol 17 (5) ◽  
pp. 784-792 ◽  
Author(s):  
R. Zichel ◽  
A. Mimran ◽  
A. Keren ◽  
A. Barnea ◽  
I. Steinberger-Levy ◽  
...  
Keyword(s):  
Expression System ◽  
Free Expression ◽  
Enzyme Linked Immunosorbent Assay ◽  
High Dose ◽  
Botulinum Toxins ◽  
Free System ◽  
Toxin Complex ◽  
A Cell ◽  
Cell Free Expression System

ABSTRACT Botulinum toxins produced by the anaerobic bacterium Clostridium botulinum are the most potent biological toxins in nature. Traditionally, people at risk are immunized with a formaldehyde-inactivated toxin complex. Second generation vaccines are based on the recombinant carboxy-terminal heavy-chain (Hc) fragment of the neurotoxin. However, the materialization of this approach is challenging, mainly due to the high AT content of clostridial genes. Herein, we present an alternative strategy in which the native genes encoding Hc proteins of botulinum toxins A, B, and E were used to express the recombinant Hc fragments in a cell-free expression system. We used the unique property of this open system to introduce different combinations of chaperone systems, protein disulfide isomerase (PDI), and reducing/oxidizing environments directly to the expression reaction. Optimized expression conditions led to increased production of soluble Hc protein, which was successfully scaled up using a continuous exchange (CE) cell-free system. Hc proteins were produced at a concentration of more than 1 mg/ml and purified by one-step Ni+ affinity chromatography. Mice immunized with three injections containing 5 μg of any of the in vitro-expressed, alum-absorbed, Hc vaccines generated a serum enzyme-linked immunosorbent assay (ELISA) titer of 105 against the native toxin complex, which enabled protection against a high-dose toxin challenge (103 to 106 mouse 50% lethal dose [MsLD50]). Finally, immunization with a trivalent HcA, HcB, and HcE vaccine protected mice against the corresponding trivalent 105 MsLD50 toxin challenge. Our results together with the latest developments in scalability of the in vitro protein expression systems offer alternative routes for the preparation of botulinum vaccine.

scholarly journals Development and Application of TK6-derived Cells Expressing Human Cytochrome P450s for Genotoxicity Testing

2020 ◽  
Vol 175 (2) ◽  
pp. 251-265 ◽  
Author(s):  
Xilin Li ◽  
Si Chen ◽  
Xiaoqing Guo ◽  
Qiangen Wu ◽  
Ji-Eun Seo ◽  
...  
Keyword(s):  
Cell Lines ◽  
Mammalian Cells ◽  
Expression System ◽  
Cytochrome P450s ◽  
Mass Spectrometry Analysis ◽  
Established Cell Line ◽  
Micronucleus Formation ◽  
Tk6 Cells ◽  
Genotoxicity Testing

Abstract Metabolism plays a key role in chemical genotoxicity; however, most mammalian cells used for in vitro genotoxicity testing lack effective metabolizing enzymes. We recently developed a battery of TK6-derived cell lines that individually overexpress 1 of 8 cytochrome P450s (CYP1A1, 1A2, 1B1, 2A6, 2B6, 2C9, 2C19, and 3A4) using a lentiviral expression system. The increased expression and metabolic function of each individual CYP in each established cell line were confirmed using real-time PCR, Western blotting, and mass spectrometry analysis; the parental TK6 cells and empty vector (EV) transduced cells had negligible CYP levels. Subsequently, we evaluated these cell lines using 2 prototypical polyaromatic hydrocarbon mutagens, 7,12-dimethylbenz[a]anthracene (DMBA) and benzo[a]pyrene (B[a]P), that require metabolic activation to exert their genotoxicity. DMBA-induced cytotoxicity, phosphorylation of histone H2A.X, and micronucleus formation were significantly increased in TK6 cells with CYP1A1, 1B1, 2B6, and 2C19 expression as compared with EV controls. B[a]P significantly increased cytotoxicity, DNA damage, and chromosomal damage in TK6 cells overexpressing CYP1A1 and 1B1 when compared with EV controls. B[a]P also induced micronucleus formation in TK6 cells expressing CYP1A2. These results suggest that our CYP-expressing TK6 cell system can be used to detect the genotoxicity of compounds requiring metabolic transformation.

scholarly journals The Plasmodium falciparum circumsporozoite protein produced in Lactococcus lactis is pure and stable

2019 ◽  
Vol 295 (2) ◽  
pp. 403-414 ◽  
Author(s):  
Susheel K. Singh ◽  
Jordan Plieskatt ◽  
Bishwanath Kumar Chourasia ◽  
Vandana Singh ◽  
Judith M. Bolscher ◽  
...  
Keyword(s):  
Plasmodium Falciparum ◽  
Lactococcus Lactis ◽  
Malaria Vaccine ◽  
Vaccine Development ◽  
Expression System ◽  
Surface Protein ◽  
Circumsporozoite Protein ◽  
Full Length

The Plasmodium falciparum circumsporozoite protein (PfCSP) is a sporozoite surface protein whose role in sporozoite motility and cell invasion has made it the leading candidate for a pre-erythrocytic malaria vaccine. However, production of high yields of soluble recombinant PfCSP, including its extensive NANP and NVDP repeats, has proven problematic. Here, we report on the development and characterization of a secreted, soluble, and stable full-length PfCSP (containing 4 NVDP and 38 NANP repeats) produced in the Lactococcus lactis expression system. The recombinant full-length PfCSP, denoted PfCSP4/38, was produced initially with a histidine tag and purified by a simple two-step procedure. Importantly, the recombinant PfCSP4/38 retained a conformational epitope for antibodies as confirmed by both in vivo and in vitro characterizations. We characterized this complex protein by HPLC, light scattering, MS analysis, differential scanning fluorimetry, CD, SDS-PAGE, and immunoblotting with conformation-dependent and -independent mAbs, which confirmed it to be both pure and soluble. Moreover, we found that the recombinant protein is stable at both frozen and elevated-temperature storage conditions. When we used L. lactis–derived PfCSP4/38 to immunize mice, it elicited high levels of functional antibodies that had the capacity to modify sporozoite motility in vitro. We concluded that the reported yield, purity, results of biophysical analyses, and stability of PfCSP4/38 warrant further consideration of using the L. lactis system for the production of circumsporozoite proteins for preclinical and clinical applications in malaria vaccine development.

scholarly journals Simultaneous monitoring of transcription and translation in mammalian cell-free expression in bulk and in cell-sized droplets

2018 ◽  
Vol 3 (1) ◽  
Author(s):  
Shue Wang ◽  
Sagardip Majumder ◽  
Nicholas J Emery ◽  
Allen P Liu
Keyword(s):  
Synthetic Biology ◽  
Real Time ◽  
Protein Dynamics ◽  
Mammalian Cell ◽  
Locked Nucleic Acid ◽  
Free Expression ◽  
Reporter Protein ◽  
Bottom Up ◽  
Lna Probe

Abstract Transcription and translation are two critical processes during eukaryotic gene expression that regulate cellular activities. The development of mammalian cell-free expression (CFE) systems provides a platform for studying these two critical processes in vitro for bottom-up synthetic biology applications such as construction of an artificial cell. Moreover, real-time monitoring of the dynamics of synthesized mRNA and protein is key to characterize and optimize gene circuits before implementing in living cells or in artificial cells. However, there are few tools for measurement of mRNA and protein dynamics in mammalian CFE systems. Here, we developed a locked nucleic acid (LNA) probe for monitoring transcription in a HeLa-based CFE system in real-time. By using this LNA probe in conjunction with a fluorescent reporter protein, we were able to simultaneously monitor mRNA and protein dynamics in bulk reactions and cell-sized single-emulsion droplets. We found rapid production of mRNA transcripts that decreased over time as protein production ensued in bulk reactions. Our results also showed that transcription in cell-sized droplets has different dynamics compared to the transcription in bulk reactions. The use of this LNA probe in conjunction with fluorescent proteins in HeLa-based mammalian CFE system provides a versatile in vitro platform for studying mRNA dynamics for bottom-up synthetic biology applications.

scholarly journals A synthetic biology platform for the reconstitution and mechanistic dissection of LINC complex assembly

2018 ◽  
Author(s):  
Sagardip Majumder ◽  
Patrick T. Willey ◽  
Maxwell S. DeNies ◽  
Allen P. Liu ◽  
G.W. Gant Luxton
Keyword(s):  
Synthetic Biology ◽  
Lipid Bilayers ◽  
Transmembrane Domain ◽  
Experimental System ◽  
Free Expression ◽  
Supported Lipid Bilayers ◽  
Linc Complex ◽  
Complex Assembly ◽  
Complex Core

ABSTRACTThe linker of nucleoskeleton and cytoskeleton (LINC) is a conserved nuclear envelope-spanning molecular bridge that is responsible for the mechanical integration of the nucleus with the cytoskeleton. LINC complexes are formed by a transluminal interaction between the outer and inner nuclear membrane KASH and SUN proteins, respectively. Despite recent structural insights, our mechanistic understanding of LINC complex assembly remains limited by the lack of an experimental system for its in vitro reconstitution and manipulation. Here, we describe artificial nuclear membranes (ANMs) as a synthetic biology platform based on mammalian cell-free expression for the rapid reconstitution of SUN proteins in supported lipid bilayers. We demonstrate that SUN1 and SUN2 are oriented in ANMs with solvent-exposed C-terminal KASH-binding SUN domains. We also find that SUN2 possesses a single transmembrane domain, while SUN1 possesses three. Finally, SUN protein-containing ANMs bind synthetic KASH peptides, thereby reconstituting the LINC complex core. This work represents the first in vitro reconstitution of KASH-binding SUN proteins in supported lipid bilayers using cell-free expression, which will be invaluable for testing proposed models of LINC complex assembly and its regulation.

scholarly journals EKPRESI ANTIGEN Ag85B DARI Mycobacterium tuberculosis PADA GALUR SEL MAMALIA

2020 ◽  
Vol 7 (1) ◽  
Author(s):  
Sabar Pambudi ◽  
Tika Widayanti ◽  
Nadya Stephanie
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Recombinant Protein ◽  
Western Blot ◽  
Mammalian Cell ◽  
Mammalian Cells ◽  
Culture Media ◽  
Mammalian Cell Culture ◽  
Expression System ◽  
Major Health Problem ◽  
Mammalian Expression

Expression of Mycobacterium tuberculosis Ag85B Antigen in Mammalian Cell CultureTuberculosis (TB) continues to be a major health problem worldwide, affecting millions of people each year. The only vaccine approved for the prevention of TB is Bacillus Calmette-Guérin (BCG). However, one of the limitations of BCG is that its preventive effect against pulmonary TB varies from person to person. Therefore, there arises a need for a new TB vaccine to replace BCG. This study aims to obtain the Ag85B recombinant protein which has characteristics similar to the native Ag85B antigen from Mycobacterium tuberculosis. In this study, we cloned and expressed recombinant Ag85B in mammalian cell culture. In the initial step, we cloned synthetic Ag85B into mammalian expression vector pFLAG-CMV4 and expressed the gene in CHO-K1 cells. Interestingly, a specific band around 30 kDa was observed in the culture media of transfected cells by Western blot analysis. The results from our research showed the potency of mammalian expression system to produce recombinant protein Ag85B for new TB vaccine candidate.Keywords: Ag85B, mammalian cells, tuberculosis, vaccine, expression ABSTRAKTuberkulosis (TB) terus menjadi salah satu masalah kesehatan dunia yang mempengaruhi jutaan manusia setiap tahun. Satu-satunya vaksin untuk TB yang ada adalah Bacillus Calmette-Guérin (BCG). Namun demikian, vaksin BCG ini memiliki kelemahan berupa terjadi efek preventif yang bervariasi dari satu individu terhadap individu lainnya.  Oleh sebab itu diperlukan pengembangan vaksin TB yang dapat menggantikan vaksin BCG yang sudah ada. Penelitian ini bertujuan memperoleh protein rekombinan Ag85B yang memiliki karakteristik mirip dengan antigen Ag85B native dari Mycobacterium tuberculosis. Pada penelitian ini, telah dilakukan kegiatan pengklonaan dan ekspresi gen Ag85B pada galur sel mamalia.  Pada tahap awal dilakukan pengklonaan gen sintesis Ag85B ke dalam plasmid pada sel mamalia pFLAG-CMV4 dan diekspresikan gennya pada sel CHO-K1. Hasil analisis Western blot menunjukan tersekresinya gen target berukuran 30 kDa pada media kultur dari sel mamalia yang ditransfeksi. Hasil dari penelitian ini menunjukkan potensi dari sistem ekpresi untuk protein rekombinan Ag85B pada galur sel mamalia sebagai kandidat vaksin TB yang baru.

scholarly journals Synthetic logic circuits using RNA aptamer against T7 RNA polymerase

2014 ◽  
Author(s):  
Jongmin Kim ◽  
Juan F Quijano ◽  
Enoch Yeung ◽  
Richard M Murray
Keyword(s):  
Rna Polymerase ◽  
Expression System ◽  
Building Blocks ◽  
Logic Circuits ◽  
T7 Rna Polymerase ◽  
Free Expression ◽  
Rna Aptamer ◽  
Cell Free Expression System

Recent advances in nucleic acids engineering introduced several RNA-based regulatory components for synthetic gene circuits, expanding the toolsets to engineer organisms. In this work, we designed genetic circuits implementing an RNA aptamer previously described to have the capability of binding to the T7 RNA polymerase and inhibiting its activity in vitro. Using in vitro transcription assays, we first demonstrated the utility of the RNA aptamer in combination with programmable synthetic transcription networks. As a step to quickly assess the feasibility of aptamer functions in vivo, a cell-free expression system was used as a breadboard to emulate the in vivo conditions of E. coli. We tested the aptamer and its three sequence variants in the cell-free expression system, verifying the aptamer functionality in the cell-free testbed. In vivo expression of aptamer and its variants demonstrated control over GFP expression driven by T7 RNA polymerase with different response curves, indicating its ability to serve as building blocks for both logic circuits and transcriptional cascades. This work elucidates the potential of RNA-based regulators for cell programming with improved controllability leveraging the fast production and degradation time scales of RNA molecules.

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