Production of Proteins prM/M and E of Dengue Virus-3 in Pichia pastoris: Simplified Purification and Evaluation of Their Use as Antigens in Serological Diagnosis of Dengue

Dengue is a major arbovirus affecting humans today. With the growing number of cases, it is essential to have large-scale production of antigens for the development of diagnostic kits for the rapid detection of patients infected by the virus and consequent proper medical intervention for them. In this work, we express the prM/M and E proteins of dengue virus-3 in yeast Pichia pastoris KM71H. The proteins were produced in soluble form in the supernatant of the culture and were purified by precipitation with ammonium sulfate. The fraction of 80% of ammonium sulfate was used as an antigen in an indirect enzyme-linked immunosorbent assay (ELISA), providing a sensitivity of 82.61% and a specificity of 89.25%. Thus, the methodology proposed here showed promise for obtaining antigens of dengue viruses and creating quick and inexpensive diagnostic tests, which is of great value since large portions of the areas affected by this disease are economically neglected.

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Large-scale production of yak (Bos grunniens) chymosin A in Pichia pastoris

Protein Expression and Purification ◽

10.1016/j.pep.2018.10.007 ◽

2019 ◽

Vol 154 ◽

pp. 126-133 ◽

Cited By ~ 4

Author(s):

Fatma Ersöz ◽

Mehmet İnan

Keyword(s):

Pichia Pastoris ◽

Large Scale ◽

Scale Production ◽

Bos Grunniens ◽

Large Scale Production

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Large-scale production, purification and bioactivity assay of recombinant human interleukin-6 in the methylotrophic yeast Pichia pastoris

FEMS Yeast Research ◽

10.1111/j.1567-1364.2010.00701.x ◽

2010 ◽

Vol 11 (2) ◽

pp. 160-167 ◽

Cited By ~ 13

Author(s):

Hongbo Li ◽

Yu Wang ◽

Aimin Xu ◽

Shiwu Li ◽

Shouguang Jin ◽

...

Keyword(s):

Pichia Pastoris ◽

Interleukin 6 ◽

Large Scale ◽

Methylotrophic Yeast ◽

Scale Production ◽

Yeast Pichia Pastoris ◽

Methylotrophic Yeast Pichia Pastoris ◽

Large Scale Production ◽

Bioactivity Assay

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Large scale production of the active human ASCT2 (SLC1A5) transporter in Pichia pastoris — functional and kinetic asymmetry revealed in proteoliposomes

Biochimica et Biophysica Acta (BBA) - Biomembranes ◽

10.1016/j.bbamem.2013.05.034 ◽

2013 ◽

Vol 1828 (9) ◽

pp. 2238-2246 ◽

Cited By ~ 41

Author(s):

Piero Pingitore ◽

Lorena Pochini ◽

Mariafrancesca Scalise ◽

Michele Galluccio ◽

Kristina Hedfalk ◽

...

Keyword(s):

Pichia Pastoris ◽

Large Scale ◽

Scale Production ◽

Large Scale Production

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Large-Scale Production of Glaciozyma antarctica Antifreeze Protein 1 (Afp1) by Fed-Batch Fermentation of Pichia pastoris

Arabian Journal for Science and Engineering ◽

10.1007/s13369-017-2738-1 ◽

2017 ◽

Vol 43 (1) ◽

pp. 133-141 ◽

Cited By ~ 2

Author(s):

Mahzan Md Tab ◽

Noor Haza Fazlin Hashim ◽

Nazalan Najimudin ◽

Nor Muhammad Mahadi ◽

Farah Diba Abu Bakar ◽

...

Keyword(s):

Pichia Pastoris ◽

Batch Fermentation ◽

Large Scale ◽

Antifreeze Protein ◽

Scale Production ◽

Fed Batch ◽

Large Scale Production ◽

Fed Batch Fermentation

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High-Level Production of a Thermostable Mutant of Yarrowia lipolytica Lipase 2 in Pichia pastoris

International Journal of Molecular Sciences ◽

10.3390/ijms21010279 ◽

2019 ◽

Vol 21 (1) ◽

pp. 279

Author(s):

Qinghua Zhou ◽

Zhixin Su ◽

Liangcheng Jiao ◽

Yao Wang ◽

Kaixin Yang ◽

...

Keyword(s):

Pichia Pastoris ◽

Yarrowia Lipolytica ◽

Large Scale ◽

Gene Dosage ◽

Life Time ◽

Industrial Applications ◽

Culture Conditions ◽

Scale Production ◽

Large Scale Production ◽

High Level

As a promising biocatalyst, Yarrowia lipolytica lipase 2 (YlLip2) is limited in its industrial applications due to its low thermostability. In this study, a thermostable YlLip2 mutant was overexpressed in Pichia pastoris and its half-life time was over 30 min at 80 °C. To obtain a higher protein secretion level, the gene dosage of the mutated lip2 gene was optimized and the lipase activity was improved by about 89%. Then, the YlLip2 activity of the obtained strain further increased from 482 to 1465 U/mL via optimizing the shaking flask culture conditions. Subsequently, Hac1p and Vitreoscilla hemoglobin (VHb) were coexpressed with the YlLip2 mutant to reduce the endoplasmic reticulum stress and enhance the oxygen uptake efficiency in the recombinant strains, respectively. Furthermore, high-density fermentations were performed in a 3 L bioreactor and the production of the YlLip2 mutant reached 9080 U/mL. The results demonstrated that the expression level of the thermostable YlLip2 mutant was predominantly enhanced via the combination of these strategies in P. pastoris, which forms a consolidated basis for its large-scale production and future industrial applications.

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NS1 Recombinant Proteins Are Efficiently Produced in Pichia pastoris and Have Great Potential for Use in Diagnostic Kits for Dengue Virus Infections

Diagnostics ◽

10.3390/diagnostics10060379 ◽

2020 ◽

Vol 10 (6) ◽

pp. 379

Author(s):

Mariana Fonseca Xisto ◽

John Willians Oliveira Prates ◽

Ingrid Marques Dias ◽

Roberto Sousa Dias ◽

Cynthia Canedo da Silva ◽

...

Keyword(s):

Pichia Pastoris ◽

Dengue Virus ◽

Recombinant Proteins ◽

Large Scale ◽

Limiting Factor ◽

Scale Production ◽

Global Public Health ◽

Virus Infections ◽

Correct Treatment ◽

Diagnostic Kits

Dengue is one of the major diseases causing global public health concerns. Despite technological advances in vaccine production against all its serotypes, it is estimated that the dengue virus is responsible for approximately 390 million infections per year. Laboratory diagnosis has been the key point for the correct treatment and prevention of this disease. Currently, the limiting factor in the manufacture of dengue diagnostic kits is the large-scale production of the non-structural 1 (NS1) antigen used in the capture of the antibody present in the infected patients’ serum. In this work, we demonstrate the production of the non-structural 1 protein of dengue virus (DENV) serotypes 1–4 (NS1-DENV1, NS1-DENV2, NS1-DENV3, and NS1-DENV4) in the methylotrophic yeast Pichia pastoris KM71H. Secreted recombinant protein was purified by affinity chromatography and characterized by SDS-PAGE and ELISA. The objectives of this study were achieved, and the results showed that P. pastoris is a good heterologous host and worked well in the production of NS1DENV 1–4 recombinant proteins. Easy to grow and quick to obtain, this yeast secreted ready-to-use proteins, with a final yield estimated at 2.8–4.6 milligrams per liter of culture. We reached 85–91% sensitivity and 91–93% specificity using IgM as a target, and for anti-dengue IgG, 83–87% sensitivity and 81–93% specificity were achieved. In this work, we conclude that the NS1 recombinant proteins are efficiently produced in P. pastoris and have great potential for use in diagnostic kits for dengue virus infections. The transformed yeast obtained can be used for production in industrial-scale bioreactors.

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Production of Human Cu,Zn SOD with Higher Activity and Lower Toxicity inE. colivia Mutation of Free Cysteine Residues

BioMed Research International ◽

10.1155/2017/4817376 ◽

2017 ◽

Vol 2017 ◽

pp. 1-12

Author(s):

Kun Zhang ◽

Yuejuan Zhang ◽

Jing Zi ◽

Xiaochang Xue ◽

Yi Wan

Keyword(s):

Large Scale ◽

Soluble Form ◽

Scale Production ◽

Superoxide Dismutase 1 ◽

Wild Type ◽

Lower Toxicity ◽

E Coli ◽

Promising Strategy ◽

Large Scale Production ◽

Cell Components

Although, as an antioxidant enzyme, human Cu,Zn superoxide dismutase 1 (hSOD1) can mitigate damage to cell components caused by free radicals generated by aerobic metabolism, large-scale manufacturing and clinical use of hSOD1 are still limited by the challenge of rapid and inexpensive production of high-quality eukaryotic hSOD1 in recombinant forms. We have demonstrated previously that it is a promising strategy to increase the expression levels of soluble hSOD1 so as to increase hSOD1 yields inE. coli. In this study, a wild-type hSOD1 (wtSOD1) and three mutant SOD1s (mhSOD1s), in which free cysteines were substituted with serine, were constructed and their expression in soluble form was measured. Results show that the substitution of Cys111 (mhSOD1/C111S) increased the expression of soluble hSOD1 inE. coliwhereas substitution of the internal Cys6 (mhSOD1/C6S) decreased it.Besides, raised levels of soluble expression led to an increase in hSOD1 yields. In addition, mhSOD1/C111S expressed at a higher soluble level showed lower toxicity and stronger whitening and antiradiation activities than those of wtSOD1. Taken together, our data demonstrate that C111S mutation in hSOD1 is an effective strategy to develop new SOD1-associated reagents and that mhSOD1/C111S is a satisfactory candidate for large-scale production.

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