scholarly journals Trends in wheat germ cell free protein expression system with an emphasis on up-scaling and industrial application

2010 ◽  
Vol 3 (3) ◽  
pp. 349-354 ◽  
Author(s):  
Bhanu Revathi K
Keyword(s):  
Protein Expression ◽  
Germ Cell ◽  
Industrial Application ◽  
Wheat Germ ◽  
Expression System ◽  
Free Protein ◽  
Protein Expression System ◽  
Up Scaling

Application of wheat germ cell-free protein expression system for novel malaria vaccine candidate discovery

2013 ◽  
Vol 13 (1) ◽  
pp. 75-85 ◽  
Author(s):  
Thangavelu U Arumugam ◽  
Daisuke Ito ◽  
Eizo Takashima ◽  
Mayumi Tachibana ◽  
Tomoko Ishino ◽  
...  
Keyword(s):  
Protein Expression ◽  
Germ Cell ◽  
Wheat Germ ◽  
Malaria Vaccine ◽  
Vaccine Candidate ◽  
Expression System ◽  
Free Protein ◽  
Protein Expression System ◽  
Malaria Vaccine Candidate

scholarly journals Expression of Recombinant Fusion Protein of Newcastle Disease Virus from Escherichia coli Plasmid Clone C-2a by In-vitro Cell-free Protein Expression System

2020 ◽  
Vol 20 ◽  
pp. 04004
Author(s):  
Ahmad Pandu Satria Wiratama ◽  
Aris Haryanto
Keyword(s):  
Protein Expression ◽  
Newcastle Disease ◽  
Recombinant Plasmid ◽  
Expression System ◽  
Free Protein ◽  
F Protein ◽  
E Coli ◽  
Protein Expression System ◽  
Sds Page

Newcastle Disease Virus (NDV) is an infectious disease that infect many kinds of wild and domesticated birds. Infection of NDV become a massive problem for poultry industry around the world especially in Indonesia. Vaccination is an effort to prevent the infection of NDV in poultry. NDV vaccine that used in Indonesia is a conventional life vaccine from LaSota and B1 strains. These type of vaccine is 21%-23% genetically distinct with the virus that spread in the environment. The antibody protection provided by the vaccine is not effective. Therefore, vaccination with new local NDV strain is needed to prevent the NDV infection in Indonesia. The previously study research reported that the local isolate of NDV from Kulon Progo, Indonesia has been isolated. Fusion (F) protein encoding gene that has been inserted into pBT7-N-His expression p lasmid which isolated from clone C-2a of E. coli, then it was expressed by the Cell-free protein expression system. The aim of this study was to confirm whether clone C-2a of E.coli carrying a recombinant plasmid pBT7-N-His-Fusion NDV and to express a recombinant F protein of NDV in-vitro from expression plasmid by cell-free protein expression system. This work started by detection of recombinant plasmid pBT7-N-His-Fusion NDV by DNA plasmid extraction followed by agarose gel electrophoresis. The recombinant F protein was in-vitro expressed by cell-free protein expression kit. The expressed F protein of NDV then was visualized by SDS-PAGE and Westernblott to analyse the expression of NDV recombinant F protein. It confirmed that clone C-2a of E. coli contained plasmid pBT7-N-His (4.001 bp) inserted by recombinant F protein of NDV gene (642 bp). The visualisation of expressed recombinant F protein by SDS-PAGE and Westernblott showed the NDV recombinant F protein was a specific protein fragment with molecular weight of 25,6 kDa..

scholarly journals A novel family of expression vectors with multiple affinity tags for wheat germ cell-free protein expression

2020 ◽  
Vol 20 (1) ◽  
Author(s):  
Szilvia Krisztina Nagy ◽  
Brigitta Margit Kállai ◽  
Judit András ◽  
Tamás Mészáros
Keyword(s):  
Protein Expression ◽  
Germ Cell ◽  
Cleavage Site ◽  
Wheat Germ ◽  
In Vitro Translation ◽  
Affinity Tags ◽  
Free Protein ◽  
Target Proteins ◽  
Interaction Studies

Abstract Background Cell-free protein expression has become a widely used alternative of in vivo, cell-based systems in functional and structural studies of proteins. The wheat germ-based method outstands from the commercially available eukaryotic in vitro translation systems by its flexibility, high translation efficiency and success rate of properly folded eukaryotic protein synthesis. The original T7 promoter containing pEU3-NII vector was improved previously by addition of a ligation-independent cloning site, His6- and GST-tags, and a TEV protease cleavage site to facilitate the creation of recombinant plasmids, permit affinity purification, and enable production of purified, tag-free target proteins, respectively. Results Here, we describe a further development of pEU3-NII vector by inserting the rare-cutting, NotI restriction enzyme cleavage site to simplify vector linearization step prior to in vitro transcription. Additionally, His12, FLAG, and Halo affinity tag coding vectors have been created to increase detection sensitivity, specificity of interaction studies, and provide covalently linkable ligands for pull-down assays, respectively. Finally, the presented GST-His6, and GST-biotin double-tagging vectors could broaden the range of possibilities of protein-protein interaction studies. Conclusions The new generation of pEU3-NII vector family allows a more rapid production of translationally active mRNA and wheat germ cell-free expression of target proteins with a wide variety of affinity tags thus enables designing flexible and diverse experimental arrangement for in vitro studies of proteins.

scholarly journals Reconstitution of Drosophila and human chromatins by wheat germ cell-free co-expression system

2020 ◽  
Vol 20 (1) ◽  
Author(s):  
Kei-ichi Okimune ◽  
Szilvia K. Nagy ◽  
Shogo Hataya ◽  
Yaeta Endo ◽  
Taichi E. Takasuka
Keyword(s):  
Protein Expression ◽  
Germ Cell ◽  
Chromatin Remodeling ◽  
Wheat Germ ◽  
Chromatin Assembly ◽  
Micrococcal Nuclease ◽  
In Vitro Translation ◽  
Free Protein ◽  
Core Histones

Abstract Background Elaboration of the epigenetic regulation of chromatin is a long-standing aim in molecular and cellular biology. Hence, there is a great demand for the development of in vitro methods to reconstitute chromatin that can be used directly for biochemical assays. The widely used wheat germ cell-free protein expression method provides broad applications to investigate the function and structure of eukaryotic proteins. Such advantages, including high translation efficiency, flexibility, and possible automatization, are beneficial for achieving native-like chromatin substrates for in vitro studies. Results We describe a novel, single-step in vitro chromatin assembly method by using the wheat germ cell-free protein synthesis. We demonstrated that both Drosophila and human chromatins can be reconstituted in the course of the in vitro translation of core histones by the addition of chromatin assembly factors, circular plasmid, and topoisomerase I in an ATP-dependent manner. Drosophila chromatin assembly was performed in 4 h at 26 °C, in the presence of premixed mRNAs encoding the core histones, dAcf1/dISWI chromatin remodeling complex, and nucleosome assembly protein, dNAP1. Similarly, the human chromatin was assembled by co-expressing the human core histones with Drosophila chromatin remodeling factor, dISWI, and chromatin chaperone, dNLP, for 6 h at 26 °C. The presence of reconstituted chromatin was monitored by DNA supercoiling assay, also the regular spacing of nucleosomes was assessed by Micrococcal nuclease assay. Furthermore, Drosophila linker histone H1-containing chromatin was reconstituted, affirming that the in vitro assembled chromatin is suitable for downstream applications. Conclusions The method described in this study allows the assembly of Drosophila and human chromatins, possibly in native-like form, by using a wheat germ cell-free protein expression. Although both chromatins were reconstituted successfully, there were unexpected differences with respect to the required ratio of histone-coding mRNAs and the reaction time. Overall, our new in vitro chromatin reconstitution method will aid to characterize the unrevealed structure, function, and regulation of chromatin dynamics.

Leishmania cell-free protein expression system

Methods ◽  
2011 ◽  
Vol 55 (1) ◽  
pp. 58-64 ◽  
Author(s):  
Oleksiy Kovtun ◽  
Sergey Mureev ◽  
WooRam Jung ◽  
Marta H. Kubala ◽  
Wayne Johnston ◽  
...  
Keyword(s):  
Protein Expression ◽  
Expression System ◽  
Free Protein ◽  
Protein Expression System

scholarly journals Easy Synthesis of Complex Biomolecular Assemblies: Wheat Germ Cell-Free Protein Expression in Structural Biology

2021 ◽  
Vol 8 ◽  
Author(s):  
Marie-Laure Fogeron ◽  
Lauriane Lecoq ◽  
Laura Cole ◽  
Matthias Harbers ◽  
Anja Böckmann
Keyword(s):  
Protein Synthesis ◽  
Protein Expression ◽  
Germ Cell ◽  
Structural Biology ◽  
Wheat Germ ◽  
New Technologies ◽  
Basic Research ◽  
Free Protein ◽  
Eukaryotic Proteins ◽  
Cell Free Protein Synthesis

Cell-free protein synthesis (CFPS) systems are gaining more importance as universal tools for basic research, applied sciences, and product development with new technologies emerging for their application. Huge progress was made in the field of synthetic biology using CFPS to develop new proteins for technical applications and therapy. Out of the available CFPS systems, wheat germ cell-free protein synthesis (WG-CFPS) merges the highest yields with the use of a eukaryotic ribosome, making it an excellent approach for the synthesis of complex eukaryotic proteins including, for example, protein complexes and membrane proteins. Separating the translation reaction from other cellular processes, CFPS offers a flexible means to adapt translation reactions to protein needs. There is a large demand for such potent, easy-to-use, rapid protein expression systems, which are optimally serving protein requirements to drive biochemical and structural biology research. We summarize here a general workflow for a wheat germ system providing examples from the literature, as well as applications used for our own studies in structural biology. With this review, we want to highlight the tremendous potential of the rapidly evolving and highly versatile CFPS systems, making them more widely used as common tools to recombinantly prepare particularly challenging recombinant eukaryotic proteins.

scholarly journals In vitro expression of the recombinant fusion protein of Newcastle disease virus from local Indonesian isolates by using a cell-free protein expression system

2020 ◽  
Vol 25 (2) ◽  
pp. 69
Author(s):  
Aris Haryanto ◽  
Hevi Wihadmadyatami ◽  
Nastiti Wijayanti
Keyword(s):  
Protein Expression ◽  
Newcastle Disease Virus ◽  
Restriction Endonuclease ◽  
Newcastle Disease ◽  
Expression System ◽  
Free Protein ◽  
F Protein ◽  
Protein Expression System ◽  
A Cell

The aim of this work was the in vitro expression of the recombinant fusion (F) protein of Newcastle disease virus (NDV).  The pBT7-N-His-Fusion-NDV expression plasmid which carries the recombinant F protein encoding gene from local Indonesian isolates, was prepared and transformed into E. coli BL21 (DE3). To detect bacterial colonies carrying the recombinant plasmid, a restriction endonuclease analysis was performed using the EcoRI restriction endonuclease. These results showed that the pBT-N-His-Fusion-NDV plasmid was successfully isolated with a size of 4.601 bp, and three recombinant plasmids carrying the gene coding for the recombinant F protein of NDV were obtained. Selected recombinant plasmids were then in vitro by using a cell-free protein expression system followed by visualization of the recombinant F protein on a 12% SDS-PAGE gel both by Coomassie Brilliant Blue staining and Western blotting. Recombinant F protein was successfully in vitro expressed by using a cell-free protein expression system as indicated by a specific single protein band with a molecular mass of 25.6 kDa.

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