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

Diagnostics ◽  
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.

scholarly journals 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

Fermentation ◽  
2020 ◽  
Vol 6 (3) ◽  
pp. 88
Author(s):  
Michelle D. O. Teixeira ◽  
Roberto S. Dias ◽  
John W. O. Prates ◽  
Juliana M. C. Monteiro ◽  
Mariana F. Xisto ◽  
...  
Keyword(s):  
Pichia Pastoris ◽  
Dengue Virus ◽  
Ammonium Sulfate ◽  
Large Scale ◽  
Medical Intervention ◽  
Soluble Form ◽  
Enzyme Linked Immunosorbent Assay ◽  
Scale Production ◽  
E Proteins ◽  
Large Scale Production

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.

scholarly journals Secretory expression of human insulin precursor in Pichia pastoris employing truncated α-factor leader sequence and a short C-peptide

2018 ◽  
Vol 23 (2) ◽  
pp. 102 ◽  
Author(s):  
Dini Nurdiani ◽  
Hariyatun Hariyatun ◽  
Wien Kusharyoto
Keyword(s):  
Pichia Pastoris ◽  
Human Insulin ◽  
Recombinant Proteins ◽  
Large Scale ◽  
Lifestyle Changes ◽  
Diabetic Patients ◽  
Scale Production ◽  
Middle Income ◽  
C Peptide ◽  
Insulin Precursor

In the past ten years, diabetes prevalence has increased rapidly in low- and middle-income countries due to lifestyle changes. This increased number of diabetic patients leads to the escalation of recombinant insulin demand, which is creating a large global insulin market. Pichia pastoris has appeared as an alternative host to produce recombinant proteins. It has excellent qualifications as an expression host for large-scale production of recombinant proteins for therapeutic use. In this study, we attempted to express the insulin precursor (IP) in P. pastoris. We used a synthetic IP-encoding gene constructed in frame with the truncated α-factor secretory signal and a short C-peptide (DGK) linked A- and B-chain of human insulin in a pD902 expression vector. Several zeocin resistant clones were successfully obtained and verified with PCR using AOX1 specific primers for the integration of the expression cassette into the P. pastoris genome and for the identification of Mut phenotypes. The secretion of IP by the Pichia pastoris clone in the culture supernatant was confirmed using SDS-PAGE, where a single band of the secreted IP with a molecular mass above 6.5 kDa was found.

Recombinant Protein Production in Microalgae: Emerging Trends

2020 ◽  
Vol 27 (2) ◽  
pp. 105-110 ◽  
Author(s):  
Niaz Ahmad ◽  
Muhammad Aamer Mehmood ◽  
Sana Malik
Keyword(s):  
Chloroplast Genome ◽  
Recombinant Proteins ◽  
Large Scale ◽  
Higher Plants ◽  
Scale Up ◽  
Chloroplast Transformation ◽  
Point Of View ◽  
Nuclear Transformation ◽  
Scale Production ◽  
Algal Chloroplast

: In recent years, microalgae have emerged as an alternative platform for large-scale production of recombinant proteins for different commercial applications. As a production platform, it has several advantages, including rapid growth, easily scale up and ability to grow with or without the external carbon source. Genetic transformation of several species has been established. Of these, Chlamydomonas reinhardtii has become significantly attractive for its potential to express foreign proteins inexpensively. All its three genomes – nuclear, mitochondrial and chloroplastic – have been sequenced. As a result, a wealth of information about its genetic machinery, protein expression mechanism (transcription, translation and post-translational modifications) is available. Over the years, various molecular tools have been developed for the manipulation of all these genomes. Various studies show that the transformation of the chloroplast genome has several advantages over nuclear transformation from the biopharming point of view. According to a recent survey, over 100 recombinant proteins have been expressed in algal chloroplasts. However, the expression levels achieved in the algal chloroplast genome are generally lower compared to the chloroplasts of higher plants. Work is therefore needed to make the algal chloroplast transformation commercially competitive. In this review, we discuss some examples from the algal research, which could play their role in making algal chloroplast commercially successful.

scholarly journals Influência da vernalização de semente na produção, crescimento e desenvolvimento de plantas de lisianthus

2018 ◽  
Vol 39 (6) ◽  
pp. 2325 ◽  
Author(s):  
Maria Yumbla-Orbes ◽  
José Geraldo Barbosa ◽  
Wagner Campos Otoni ◽  
Marcel Santos Montezano ◽  
José Antônio Saraiva Grossi ◽  
...  
Keyword(s):  
Large Scale ◽  
Limiting Factor ◽  
Scale Production ◽  
Cut Flower ◽  
Cut Flowers ◽  
Flower Production ◽  
Commercial Scale ◽  
Large Scale Production ◽  
And Control

Flowering induction and control is a limiting factor when commercially producing cut flowers of lisianthus and seed exposure to low temperatures, a physiological event called vernalization, induces the differentiation of vegetative buds to reproductive buds, contributing to a flowering that is uniform and has quality. The objective of this study was to evaluate the influence of seed vernalization in three cultivars of lisianthus (Excalibur, Echo and Mariachi) for 12, 24, 36 and 48 days at temperatures of 5, 10 and 15°C, in the production and quality of buds, making this technology feasible to large-scale production. During cultivation it was observed that the lower the temperature and higher the vernalization period, the lower the cycle and the greater the number of plants induced to flowering for all three cultivars, and those are important features in the context of flower production in a commercial scale. The seeds subjected to vernalization originated plants that produce flower stems within the standards required by the market, showing that vernalization was efficient to induce flowering without affecting the quality of the buds. To produce lisianthus as a cut flower of quality, it is recommended seed vernalization of Mariachi and Echo cultivars for 24 days at 5°C and Excalibur for 36 days at 5°C.

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

2013 ◽  
Vol 1828 (9) ◽  
pp. 2238-2246 ◽  
Author(s):  
Piero Pingitore ◽  
Lorena Pochini ◽  
Mariafrancesca Scalise ◽  
Michele Galluccio ◽  
Kristina Hedfalk ◽  
...  
Keyword(s):  
Pichia Pastoris ◽  
Large Scale ◽  
Scale Production ◽  
Large Scale Production

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

2017 ◽  
Vol 43 (1) ◽  
pp. 133-141 ◽  
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

scholarly journals High-level expression of an acidic thermostable xylanase in Pichia pastoris and its application in weaned piglets

2019 ◽  
Vol 98 (1) ◽  
Author(s):  
Jian Wang ◽  
Yajing Liu ◽  
Yongzhi Yang ◽  
Chengling Bao ◽  
Yunhe Cao
Keyword(s):  
Pichia Pastoris ◽  
Growth Performance ◽  
Large Scale ◽  
Animal Feed ◽  
Nutrient Digestibility ◽  
Control Group ◽  
Scale Production ◽  
Thermostable Xylanase ◽  
Weaned Piglets ◽  
Degrading Bacteria

Abstract An acidic thermostable xylanase (AT-xynA) which was stable at low pH and high temperature was considered to have great potential in animal feed. For large-scale production, AT-xynA activity was enhanced about 1-fold in Pichia pastoris by constructing a double-copy expression strain in this study. Furthermore, impacts of different AT-xynA levels on growth performance, nutrient digestibility, short-chain fatty acids, and bacterial community in weaned piglets were determined. Compared with the control group, ADFI and ADG were higher for the pigs fed 4,000 or 6,000 U/kg AT-xynA (P < 0.05). AT-xynA supplementation also significantly increased the digestibility of OM, GE, and DM (P < 0.05). AT-xynA supplementation increased the concentrations of acetate in ileal (P < 0.01) and cecal digesta (P < 0.05). Isobutyrate (P < 0.05) and valerate (P < 0.05) concentrations in colonic digesta also significantly increased compared with the control group. AT-xynA supplementation increased the abundance of Lactobacillus in the ileal, cecal, and colonic digesta of weaned piglets (P < 0.05). AT-xynA alleviated anti-nutritional effects of nonstarch polysaccharides (NSP) by preventing the growth of Pateurella and Leptotrichia in the ileum (P < 0.05). AT-xynA increased the abundance of NSP-degrading bacteria, such as Ruminococcaceae, Prevotella in the cecum and colon (P < 0.05). In summary, AT-xynA addition could improve the growth performance of weaned piglets by altering gut microbiota.

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

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.

Sign in / Sign up

Export Citation Format

Share Document