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.

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..

A Novel Method for Producing Custom-Made Idiotype Vaccines for Lymphoma Immunotherapy Using a Cell-Free Expression System.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 1410-1410
Author(s):  
Gregg Kanter ◽  
Junhao Yang ◽  
Alexei Voloshin ◽  
James R. Swartz ◽  
Ronald Levy
Keyword(s):  
Amino Acid ◽  
Fusion Protein ◽  
Protein Expression ◽  
Expression System ◽  
B Cell Lymphoma ◽  
Free System ◽  
Free Protein ◽  
Protein Expression System ◽  
Custom Made ◽  
A Cell

Abstract Idiotype vaccines have been used as a customized immunotherapy for lymphoma. This strategy takes advantage of a unique antigen (either immunoglobulin or T cell receptor) found on the lymphoma cells of each patient. To make this personalized vaccine feasible for widespread use a production method, which is rapid and relatively inexpensive will be required. In an E. coli-based cell-free protein expression system we were able to make ScFv proteins derived from the immunoglobulins of a mouse B cell lymphoma, 38C13, in high yields. We have used this cell-free system to make 38C13 ScFv proteins fused to either the cytokine mGM-CSF or a 9 amino acid peptide derived from hIL-1b. The 9 amino acid fragment of hIL-1b was chosen because it is known to retain immunostimulatory function while lacking pyrogenicity. We were able consistently to produce these proteins in high yield and to purify them using a histadine tag at the terminus of the molecule. The ScFv portion of the molecule is folded properly, as assayed by its binding to an anti-idiotype Ab. Similarly, the mGM-CSF fused to the ScFv can be recognized by an anti-mGM-CSF Ab. The mGM-CSF portion is biologically active as determined by its ability to support the growth a mGM-CSF dependant cell line. We immunized C3H mice with either the ScFc-hIL-1b or the ScFv-mGM-CSF fusion protein and we were able to induce an idiotype-specific Ab response, which recognizes the native idiotype protein in ELISA assays as well as the 38C13 tumor as detected by flow cytometry. Most importantly, in the highly aggressive 38C13 lymphoma model system, three bi-weekly vaccinations with either fusion protein protected mice from tumor challenge with the syngeneic 38C13 tumor. These data establish that a cell-free protein expression system can work for producing individualized idiotype vaccines for lymphoma. (This work was supported by NIH grant CA34233)

scholarly journals Newcastle Disease Virus Establishes Persistent Infection in Tumor Cells In Vitro: Contribution of the Cleavage Site of Fusion Protein and Second Sialic Acid Binding Site of Hemagglutinin-Neuraminidase

2017 ◽  
Vol 91 (16) ◽  
Author(s):  
Udaya S. Rangaswamy ◽  
Weijia Wang ◽  
Xing Cheng ◽  
Patrick McTamney ◽  
Danielle Carroll ◽  
...  
Keyword(s):  
Newcastle Disease Virus ◽  
Disease Virus ◽  
Receptor Binding ◽  
Newcastle Disease ◽  
Persistent Infection ◽  
Cleavage Site ◽  
Protein Cleavage ◽  
F Protein ◽  
Sialic Acid Binding

ABSTRACT Newcastle disease virus (NDV) is an oncolytic virus being developed for the treatment of cancer. Following infection of a human ovarian cancer cell line (OVCAR3) with a recombinant low-pathogenic NDV, persistent infection was established in a subset of tumor cells. Persistently infected (PI) cells exhibited resistance to superinfection with NDV and established an antiviral state, as demonstrated by upregulation of interferon and interferon-induced genes such as myxoma resistance gene 1 (Mx1) and retinoic acid-inducing gene-I (RIG-I). Viruses released from PI cells induced higher cell-to-cell fusion than the parental virus following infection in two tumor cell lines tested, HT1080 and HeLa, and remained attenuated in chickens. Two mutations, one in the fusion (F) protein cleavage site, F117S (F117S), and another in hemagglutinin-neuraminidase (HN), G169R (HN169R), located in the second sialic acid binding region, were responsible for the hyperfusogenic phenotype. F117S improves F protein cleavage efficiency, facilitating cell-to-cell fusion, while HN169R possesses a multifaceted role in contributing to higher fusion, reduced receptor binding, and lower neuraminidase activity, which together result in increased fusion and reduced viral replication. Thus, establishment of persistent infection in vitro involves viral genetic changes that facilitate efficient viral spread from cell to cell as a potential mechanism to escape host antiviral responses. The results of our study also demonstrate a critical role in the viral life cycle for the second receptor binding region of the HN protein, which is conserved in several paramyxoviruses. IMPORTANCE Oncolytic Newcastle disease virus (NDV) could establish persistent infection in a tumor cell line, resulting in a steady antiviral state reflected by constitutively expressed interferon. Viruses isolated from persistently infected cells are highly fusogenic, and this phenotype has been mapped to two mutations, one each in the fusion (F) and hemagglutinin-neuraminidase (HN) proteins. The F117S mutation in the F protein cleavage site improved F protein cleavage efficiency while the HN169R mutation located at the second receptor binding site of the HN protein contributed to a complex phenotype consisting of a modest increase in fusion and cell killing, lower neuraminidase activity, and reduced viral growth. This study highlights the intricate nature of these two mutations in the glycoproteins of NDV in the establishment of persistent infection. The data also shed light on the critical balance between the F and HN proteins required for efficient NDV infection and their role in avian pathogenicity.

scholarly journals Evidence for Mixed Membrane Topology of the Newcastle Disease Virus Fusion Protein

2003 ◽  
Vol 77 (3) ◽  
pp. 1951-1963 ◽  
Author(s):  
Lori W. McGinnes ◽  
Julie N. Reitter ◽  
Kathy Gravel ◽  
Trudy G. Morrison
Keyword(s):  
Fusion Protein ◽  
Newcastle Disease Virus ◽  
Disease Virus ◽  
Newcastle Disease ◽  
Cytoplasmic Domain ◽  
Classical Type ◽  
F Protein ◽  
Amino Terminal ◽  
Protease Digestion ◽  
A Cell

ABSTRACT The synthesis of the Newcastle disease virus (NDV) fusion (F) protein in a cell-free protein-synthesizing system containing membranes was characterized. The membrane-associated products were in at least two different topological forms with respect to the membranes. The properties of one form were consistent with the expected membrane insertion as a classical type 1 glycoprotein. This form of the protein was fully glycosylated, and sequences amino terminal to the transmembrane domain were protected from protease digestion by the membranes. The second form of membrane-associated F protein was partially glycosylated and partially protected from protease digestion by the membranes. Protease digestion resulted in a 23-kDa protease-protected polypeptide derived from F2 sequences and sequences from the amino-terminal end of the F1 domain. Furthermore, a 10-kDa polypeptide derived from the cytoplasmic domain (CT) was also protected from protease digestion by the membranes. Protease resistance of the 23- and 10-kDa polypeptides suggested that this second form of F protein inserted in membranes in a polytopic conformation with both the amino-terminal end and the carboxyl-terminal end translocated across membranes. To determine if this second form of the fusion protein could be found in cells expressing the F protein, two different approaches were taken. A polypeptide with the size of the partially translocated F protein was detected by Western analysis of proteins in total-cell extracts of NDV strain B1 (avirulent)-infected Cos-7 cells. Using antibodies raised against a peptide with sequences from the cytoplasmic domain, CT sequences were detected on surfaces of F protein-expressing Cos-7 cells by immunofluorescence and by flow cytometry. This antibody also inhibited the fusion of red blood cells to cells expressing F and HN proteins. These results suggest that NDV F protein made both in a cell-free system and in Cos-7 cells may exist in two topological forms with respect to membranes and that the second form of the protein may be involved in cell-cell fusion.

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
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