Production of recombinant dengue non-structural 1 (NS1) proteins from clinical virus isolates

Yohan, Benediktus; Wardhani, Puspa; Aryati,; Trimarsanto, Hidayat; Sasmono, R. Tedjo

doi:10.1016/j.pep.2016.09.007

Cited by 5 publications

(7 citation statements)

References 27 publications

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“…Several authors had proposed the use of prokaryotic systems for high-level expression of DENV NS1 (Das et al, 2009;Sankar et al, 2013;Yohan et al, 2017;Chura-Chambi et al, 2019). However, this system often leads to the loss of conformational epitopes, since it is well described that E. coli expressed proteins tend to precipitate under the form of inclusion bodies (Das et al, 2009;Sankar et al, 2013).…”

Section: Discussionmentioning

confidence: 99%

“…The production of recombinant NS1 protein in Escherichia coli often leads to insoluble, aggregated, or incorrectly folded products (Das et al, 2009;Sankar et al, 2013;Yohan et al, 2017;Chura-Chambi et al, 2019), which may compromise their utility in immunodiagnostic tests (Peternel et al, 2011). Different expression vectors have been developed to circumvent these limitations, including mammalian cells, insect cells, yeasts, and transgenic plants (Avirutnan et al, 2006;Nascimento et al, 2018;Altmann et al, 1999;Amaro et al, 2015;Thiemmeca et al, 2016;Allonso et al, 2019).…”

Section: Introductionmentioning

confidence: 99%

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Transient Expression of Dengue Virus NS1 Antigen in Nicotiana benthamiana for Use as a Diagnostic Antigen

et al. 2020

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Dengue is a viral disease that represents a significant threat to global public health since billions of people are now at risk of infection by this mosquito-borne virus. The implementation of extensive screening tests is indispensable to control this disease, and the Dengue virus non-structural protein 1 (NS1) is a promising antigen for the serological diagnosis of dengue fever. Plant-based systems can be a safe and costeffective alternative for the production of dengue virus antigens. In this work, two strategies to produce the dengue NS1 protein in Nicotiana benthamiana leaves were evaluated: Targeting NS1 to five different subcellular compartments to assess the best subcellular organelle for the expression and accumulation of NS1, and the addition of elastin-like polypeptide (ELP) or hydrophobin (HFBI) fusion tags to NS1. The transiently expressed proteins in N. benthamiana were quantified by Western blot analysis. The NS1 fused to ELP and targeted to the ER (NS1 ELP-ER) showed the highest yield (445 mg/kg), approximately a forty-fold increase in accumulation levels compared to the non-fused protein (NS1-ER), representing the first example of transient expression of DENV NS1 in plant. We also demonstrated that NS1 ELP-ER was successfully recognized by a monoclonal anti-dengue virus NS1 glycoprotein antibody, and by sera from dengue virus-infected patients. Interestingly, it was found that transient production of NS1-ER and NS1 ELP-ER using vacuum infiltration of whole plants, which is easier to scale up, rather than syringe infiltration of leaves, greatly improved the accumulation of NS1 proteins. The generated plant made NS1, even without extensive purification, showed potential to be used for the development of the NS1 diagnostic tests in resource-limited areas where dengue is endemic.

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Transient Expression of Dengue Virus NS1 Antigen in Nicotiana benthamiana for Use as a Diagnostic Antigen

et al. 2020

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“…Inclusion bodies were solubilized by combining the method described previously by Athmaram et al (2013) and Yohan et al (2017) with some modifications. Washed inclusion bodies were added with 1:7 unfold buffer (50 mM Tris-Cl pH 8, 100 mM NaCl, 11 mM dithiothreitol, 8 M urea) and suspension was sonicated with frequency of 4,000 Hz for 5 min on (15 s on and 45 s off).…”

Section: Isolation and Refolding Of Denv-2 Ns1 Proteinmentioning

confidence: 99%

“…5). Though some differences were observed on the second and third epitopes (127)(128)(129)(130)(131)(132)(133)(134)(135)(136)(137)(138)(139)(140)(141)(142)(143)(193)(194)(195)(196)(197)(198)(199)(200)(201)(202)(203)(204)(205)(206)(207)(208)(209), the recombinant proteins were all immunogenic (Yohan et al, 2017;Lemos et al, 2013;Rozen-Gagnon et al, 2012;Zhou et al, 2006). Out of the 3 conformational epitopes, the second epitope, 127 ELHNQTFLIDGPETAEC 143 , was reported as an immunodominant B cell epitopes in DENV2-NS1 protein (Jiang et al, 2010).…”

Section: The Refolded Untagged Denv-2 Ns1 Interacts With Anti Ns1 Antmentioning

confidence: 99%

“…NS1 is secreted into blood circulation in a few days after infection and can achieve high circulating level (≥600 ng/mL) within 72 h of illness (Libraty et al, 2002), making this protein a potential biomarker for early dengue infection detection. Among all four dengue serotypes (DENV-1, DENV-2, DENV-3 and DENV-4), DENV-2 was frequently associated with most severe cases and epidemics (Soo et al, 2016;Yung et al, 2015;Vicente et al, 2016) Expression of DENV-2 NS1 from different origins has been reported in various host cells ranging from prokaryotic cells, such as E. coli BL21 (DE3) (Yohan et al, 2017) and E. coli Rosetta (DE3) (Lemos et al, 2013), to eukaryotic cells, such as Pichia pastoris X-33 (Zhou et al, 2006), P. pastoris KM71 (Puspasari et al, 2017) and insect cells (Rozen-Gagnon et al, 2012). All of the recombinant proteins, however, had a different solubility depends on the host cells in which it was expressed.…”

Section: Introductionmentioning

confidence: 99%

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Production of Immunologically Active Untagged Recombinant DENV-2 NS1 in Escherichia coli

Natalia

Heryakusuma

Puspasari

et al. 2018

American Journal of Biochemistry and Biotechnology

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Dengue is an infectious disease affecting 390 millions people in tropical and subtropical region annually. Dengue virus NS1 protein plays an important role in viral replication in the host cell and it is detected in high level in the infected patient serum. A synthetic gene of untagged DENV-2 NS1 with codons optimization for expression in Escherichia coli has been generated and then inserted into an expression vector pET16b. As a hydrophobic protein with aliphatic index of 71.21%, DENV-2 NS1 was produced as inclusion bodies in E. coli BL21(DE3). The DENV-2 NS1 aggregate was unfolded in 8 M urea and then refolded by reverse dilution method. The refolded DENV-2 NS1 has immunologically active structure as it is capable to interact with anti-NS1 antibody, hence making it as a potential vaccine candidate.

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Production of recombinant NS1 protein and its possible use in encephalitic flavivirus differential diagnosis

Lorch

Collado

Argüelles

et al. 2019

Protein Expression and Purification

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Production of recombinant dengue non-structural 1 (NS1) proteins from clinical virus isolates

Cited by 5 publications

References 27 publications

Transient Expression of Dengue Virus NS1 Antigen in Nicotiana benthamiana for Use as a Diagnostic Antigen

Transient Expression of Dengue Virus NS1 Antigen in Nicotiana benthamiana for Use as a Diagnostic Antigen

Production of Immunologically Active Untagged Recombinant DENV-2 NS1 in Escherichia coli

Production of recombinant NS1 protein and its possible use in encephalitic flavivirus differential diagnosis

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