Tick-borne encephalitis nonstructural protein NS1 expressed in E. coli retains immunological properties of the native protein

2022 ◽  
Vol 191 ◽  
pp. 106031
Author(s):  
Matveev Andrey ◽  
Khlusevich Yana ◽  
Golota Olga ◽  
Kravchuk Bogdana ◽  
Tkachev Sergey ◽  
...  
2019 ◽  
Vol 20 (18) ◽  
pp. 4416 ◽  
Author(s):  
Lara Console ◽  
Maria Tolomeo ◽  
Matilde Colella ◽  
Maria Barile ◽  
Cesare Indiveri

Background: the SLC52A2 gene encodes for the riboflavin transporter 2 (RFVT2). This transporter is ubiquitously expressed. It mediates the transport of Riboflavin across cell membranes. Riboflavin plays a crucial role in cells since its biologically active forms, FMN and FAD, are essential for the metabolism of carbohydrates, amino acids, and lipids. Mutation of the Riboflavin transporters is a risk factor for anemia, cancer, cardiovascular disease, neurodegeneration. Inborn mutations of SLC52A2 are associated with Brown-Vialetto-van Laere syndrome, a rare neurological disorder characterized by infancy onset. In spite of the important metabolic and physio/pathological role of this transporter few data are available on its function and regulation. Methods: the human recombinant RFVT2 has been overexpressed in E. coli, purified and reconstituted into proteoliposomes in order to characterize its activity following the [3H]Riboflavin transport. Results: the recombinant hRFVT2 showed a Km of 0.26 ± 0.07 µM and was inhibited by lumiflavin, FMN and Mg2+. The Riboflavin uptake was also regulated by Ca2+. The native protein extracted from fibroblast and reconstituted in proteoliposomes also showed inhibition by FMN and lumiflavin. Conclusions: proteoliposomes represent a suitable model to assay the RFVT2 function. It will be useful for screening the mutation of RFVT2.


2016 ◽  
Vol 50 (2) ◽  
pp. 307-312
Author(s):  
Y. V. Kuzmenko ◽  
E. S. Starodubova ◽  
G. G. Karganova ◽  
A. V. Timofeev ◽  
V. L. Karpov

2010 ◽  
Vol 167 (2) ◽  
pp. 186-192 ◽  
Author(s):  
Jaime Henrique Amorim ◽  
Bruna F.M.M. Porchia ◽  
Andrea Balan ◽  
Rafael C.M. Cavalcante ◽  
Simone Morais da Costa ◽  
...  

1985 ◽  
Vol 162 (5) ◽  
pp. 1720-1725 ◽  
Author(s):  
A Yamada ◽  
J F Young ◽  
F A Ennis

We have tested the ability of the c13 protein, which is a hybrid protein of the first 81 amino acids of the viral nonstructural protein (NS1) and the HA2 subunit of viral hemagglutination produced in E. coli, to render target cells susceptible to the lytic activity of influenza virus-specific cytotoxic T lymphocytes (CTL). The results showed that P815 cells coated with c13 protein were lysed by PR8 virus-induced secondary CTL derived from BALB/c mice. Cold-target inhibition tests clearly demonstrated that c13 protein-coated P815 cells were recognized by an H1 subtype-specific CTL population. Furthermore, PR8 virus-induced CTL derived from C3H mice did not lyse c13 protein-coated P815 cells, suggesting that c13 protein was recognized by CTL in conjunction with H-2d products. These findings suggest that this protein interacts with the cellular plasma membrane and makes target cells recognizable by H-2-restricted, influenza virus subtype-specific CTL.


1962 ◽  
Vol 45 (4) ◽  
pp. 125-141 ◽  
Author(s):  
John J. Hutton ◽  
Walther F. Goebel

Colicine V has been obtained from the culture medium in which the colicinogenic bacillus E. coli K357 L_T is grown. The material is electrophoretically homogeneous and proves to be a lipocarbohydrate protein complex identical with the type-specific O antigen of the parent bacillus. Colicine V is toxic both for mice and for rabbits and readily stimulates the elaboration of precipitins and bacterial agglutinins, as well as antibodies which neutralize the antibacterial activity of the colicine itself. The colicine is also toxic for certain strains of Enterobacteriaceae. Although colicine V and colicine K, previously described in this laboratory, have many properties in common, they exhibit no cross-serological relationship whatsoever.


2006 ◽  
Vol 80 (3) ◽  
pp. 1340-1351 ◽  
Author(s):  
Kyung Min Chung ◽  
Grant E. Nybakken ◽  
Bruce S. Thompson ◽  
Michael J. Engle ◽  
Anantha Marri ◽  
...  

ABSTRACT The flavivirus nonstructural protein NS1 is a highly conserved secreted glycoprotein that does not package with the virion. Immunization with NS1 elicits a protective immune response against yellow fever, dengue, and tick-borne encephalitis flaviviruses through poorly defined mechanisms. In this study, we purified a recombinant, secreted form of West Nile virus (WNV) NS1 glycoprotein from baculovirus-infected insect cells and generated 22 new NS1-specific monoclonal antibodies (MAbs). By performing competitive binding assays and expressing truncated NS1 proteins on the surface of yeast (Saccharomyces cerevisiae) and in bacteria, we mapped 21 of the newly generated MAbs to three NS1 fragments. Prophylaxis of C57BL/6 mice with any of four MAbs (10NS1, 14NS1, 16NS1, and 17NS1) strongly protected against lethal WNV infection (75 to 95% survival, respectively) compared to saline-treated controls (17% survival). In contrast, other anti-NS1 MAbs of the same isotype provided no significant protection. Notably, 14NS1 and 16NS1 also demonstrated marked efficacy as postexposure therapy, even when administered as a single dose 4 days after infection. Virologic analysis showed that 17NS1 protects at an early stage in infection through a C1q-independent and Fc γ receptor-dependent pathway. Interestingly, 14NS1, which maps to a distinct region on NS1, protected through a C1q- and Fc γ receptor-independent mechanism. Overall, our data suggest that distinct regions of NS1 can elicit protective humoral immunity against WNV through different mechanisms.


1989 ◽  
Vol 264 (2) ◽  
pp. 533-538 ◽  
Author(s):  
E A Cook ◽  
J P Huggins ◽  
G Sathe ◽  
P J England ◽  
J R Piggott

A synthetic phospholamban gene has been cloned and expressed in Escherichia coli, producing both native phospholamban and a fusion protein with 81 amino acids of the influenza virus NS1 protein. Both the native phospholamban and fusion proteins produced extensive cell lysis upon induction of gene expression, but only the native protein underwent spontaneous pentamer formation in E. coli. Translation in vitro of mRNA produced by transcription in vitro of phospholamban cDNA was used to demonstrate the spontaneous aggregation of phospholamban to form pentamers in this system also, both in the presence and absence of exogenous microsomes from canine pancreas or heart. Phospholamban produced by translation in vitro was apparently susceptible to proteolysis by enzymes present in the particulate fractions in these experiments.


2020 ◽  
Vol 58 (4) ◽  
Author(s):  
P. Girl ◽  
M. Bestehorn-Willmann ◽  
S. Zange ◽  
J. P. Borde ◽  
G. Dobler ◽  
...  

ABSTRACT Tick-borne encephalitis virus (TBEV) is an important central nervous system (CNS) infection in Europe and Asia. It is a flavivirus in the tick-borne group. Effective vaccines against TBE are available in the affected countries. However, diagnosing TBE is challenging due to cross-reactive antibodies between different viruses of the genus Flavivirus, family Flaviviridae. Differentiation between infection-induced and vaccine-induced antibodies can be difficult and in many cases impossible, due to the increasing vaccination rate against TBEV. We present a new approach to detect antibodies against the TBEV nonstructural protein 1 (NS1) as a diagnostic marker, which is exclusively indicative for virus replication in natural infection, on the basis of an enzyme-linked immunosorbent assay (ELISA). A total of 188 anonymous serum samples from the National Consultant Laboratory for TBEV were included in our study. The assay was validated according to the European Laboratory Norm DIN EN ISO 15189 for diagnostic use. The ELISA for the detection of TBEV NS1 specific IgG class antibodies has demonstrated a sensitivity of >94% and a specificity of >93% in broadly cross-reacting sera from patients with vaccinations against flaviviral diseases and single or multiple flavivirus infections, respectively. The detection of anti-NS1 antibodies is feasible and facilitates reliable differentiation between different flavivirus infections, TBEV infection, and TBE vaccination.


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