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 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.

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

Mannanase Man23 mutant library construction based on a novel cell-free protein expression system

2016 ◽  
Vol 97 (7) ◽  
pp. 2199-2204 ◽  
Author(s):  
Haiyan Zhou ◽  
Jie Yong ◽  
Han Gao ◽  
Tong Li ◽  
Hongshi Xiao ◽  
...  
Keyword(s):  
Protein Expression ◽  
Expression System ◽  
Mutant Library ◽  
Free Protein ◽  
Library Construction ◽  
Protein Expression System

scholarly journals Expression comparison between two genes encoding CSF3 recombinant proteins having different codon composition at N-terminal in Escherichia coli

2021 ◽  
Vol 948 (1) ◽  
pp. 012081
Author(s):  
K S Dewi ◽  
F D Wahyuni ◽  
S Salsabila ◽  
Aminah ◽  
N D Yanthi ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Protein Expression ◽  
Recombinant Proteins ◽  
Recombinant Plasmid ◽  
Synonymous Codon ◽  
Expression System ◽  
Negative Control ◽  
Translation Initiation Region ◽  
E Coli ◽  
Genes Encoding

Abstract Colony-stimulating factor 3 (CSF3) is a glycoprotein with many therapeutic applications. In the Escherichia coli expression system, mRNA folding and stability near the translation initiation region (TIR) are known to influence protein expression significantly. We have successfully constructed the recombinant plasmid carrying genes encoding CSF3.1 and CSF3.2, which have different synonymous codon usage at N-terminal. In this study, we compared both expressions of CSF3.1 and CSF3.2 recombinant proteins in E. coli host. Recombinant plasmid pJ414-CSF3.1 and pJ414-CSF3.2 were transformed individually into E. coli NiCo21(DE3) competent cells by a heat-shock method, then spread on solid Lysogeny Broth (LB) medium containing ampicillin. Eight transformant colonies were selected and then expressed in 2xYT medium with the addition of IPTG inducer. Expression analysis was carried out using 15% SDS-PAGE gel. No significantly different band was observed in CSF3.1 protein expression compared to the negative control. In contrast, CSF3.2 protein can be expressed with a good amount at its expected size of 18 kDa. This result was strengthened by bioinformatics analysis which demonstrated the more open TIR of CSF3.2 than that of CSF3.1 Our study highlighted that AU-rich mRNA at the N-terminal is essential for efficient recognition of the ribosome binding site.

scholarly journals Akaby - cell-free protein expression system for linear templates

2021 ◽  
Author(s):  
Wakana Sato ◽  
Judee Sharon ◽  
Christopher Deich ◽  
Nathaniel Gaut ◽  
Brock Cash ◽  
...  
Keyword(s):  
Protein Expression ◽  
Expression System ◽  
Nuclease Activity ◽  
Coli Strain ◽  
Free Expression ◽  
Dna Templates ◽  
Free Protein ◽  
E Coli ◽  
Linear Dna ◽  
Simple Alternative

Cell-free protein expression is increasingly becoming popular for biotechnology, biomedical and research applications. Among cell-free systems, the most popular one is based on Escherichia coli (E. coli). Endogenous nucleases in E. coli cell-free transcription-translation (TXTL) degrade the free ends of DNA, resulting in inefficient protein expression from linear DNA templates. RecBCD is a nuclease complex that plays a major role in nuclease activity in E. coli, with the RecB subunit possessing the actual nuclease activity. We created a RecB knockout of an E. coli strain optimized for cell-free expression. We named this new strain Akaby. We demonstrated that Akaby TXTL successfully reduced linear DNA degradations, rescuing the protein expression efficiency from the linear DNA templates. The practicality of Akaby for TXTL is an efficient, simple alternative for linear template expression in cell-free reactions. We also use this work as a model protocol for modifying the TXTL source E. coli strain, enabling the creation of TXTL systems with other custom modifications.

Construction of homo- and heteropolymers of plant ferritin subunits using an in vitro protein expression system

2007 ◽  
Vol 56 (2) ◽  
pp. 237-246 ◽  
Author(s):  
Taro Masuda ◽  
Fumiyuki Goto ◽  
Toshihiro Yoshihara ◽  
Toru Ezure ◽  
Takashi Suzuki ◽  
...  
Keyword(s):  
Protein Expression ◽  
Expression System ◽  
Protein Expression System ◽  
In Vitro Protein Expression ◽  
Vitro Protein
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