scholarly journals Production of Human Pancreatitis-Associated Protein (hPAP) in Komagataella phaffii (Pichia pastoris)

2021 ◽  
Vol 5 (2) ◽  
pp. 31
Author(s):  
Didem Pazarli ◽  
Fatıma Yücel ◽  
Esin Akçael ◽  
Şerife Şeyda Pirinçci Göktürk
Keyword(s):  
Pichia Pastoris ◽  
Early Stage ◽  
Sandwich Elisa ◽  
Stress Protein ◽  
Biological Marker ◽  
Pancreatic Acinar Cells ◽  
Efficient Production ◽  
Methanol Induction ◽  
Komagataella Phaffii ◽  
Pancreatitis Associated Protein

Pancreatitis-associated protein (PAP) is a pancreatic stress protein that is not produced in a healthy pancreas but is highly synthesized in pancreatic acinar cells in response to acute and chronic pancreatitis, hypoxia, toxins, diabetes, lipopolysaccharides hypotransferrinemia and organ transplantation. Changes in the PAP levels in serum are an important biological marker in the early stage of pancreatic diseases. In this study, the recombinant human PAP protein, which has the potential to be used as a diagnostic marker and as research material in proliferation, apoptosis, cell migration, cell invasion, and immunoassay studies, was expressed efficiently under the control of the AOX1 gene promoter in the Komagataella phaffii (Pichia pastoris) (K. phaffii) X33 strain. We describe the conditions required for the efficient production of PAP protein by methanol induction and its use without purification. The produced unpurified protein was tested in sandwich ELISA and showed consistent results with the commercial product. These results are encouraging that the protein produced can be used as a biomarker standard in ELISA tests without the cost and labor of purification.

Methanol Induction Optimization for Recombinant Human Consensus Interferon Mutant Production in Pichia pastoris

2010 ◽  
Vol 150 ◽  
pp. 540-540
Author(s):  
Dan Wu ◽  
Ju Chu ◽  
Yu-You Hao ◽  
Yong-Hong Wang ◽  
Ying-Ping Zhuang ◽  
...  
Keyword(s):  
Pichia Pastoris ◽  
Methanol Induction ◽  
Consensus Interferon

Effect of two preservation methods on the viability and enzyme production of a recombinant Komagataella phaffii (Pichia pastoris) strain

Cryobiology ◽  
2021 ◽  
Author(s):  
Alvarado-Fernández Angela María ◽  
Edwin Alexander Rodríguez-López ◽  
Angela Johana Espejo-Mojica ◽  
Angela Rocío Mosquera-Arévalo ◽  
Carlos Javier Alméciga-Díaz ◽  
...  
Keyword(s):  
Pichia Pastoris ◽  
Enzyme Production ◽  
Pastoris Strain ◽  
Preservation Methods ◽  
Komagataella Phaffii

scholarly journals Improved Production ofAspergillus usamiiendo-β-1,4-Xylanase inPichia pastorisvia Combined Strategies

2016 ◽  
Vol 2016 ◽  
pp. 1-9 ◽  
Author(s):  
Jianrong Wang ◽  
Yangyuan Li ◽  
Danni Liu
Keyword(s):  
Pichia Pastoris ◽  
Cell Viability ◽  
Recombinant Proteins ◽  
Recombinant Strain ◽  
Xylanase Activity ◽  
Vitreoscilla Hemoglobin ◽  
Wild Type ◽  
Methanol Induction ◽  
Aspergillus Usamii ◽  
Induction Temperature

A series of strategies were applied to improve expression level of recombinant endo-β-1,4-xylanase fromAspergillus usamii(A. usamii) inPichia pastoris(P. pastoris). Firstly, the endo-β-1,4-xylanase (xynB) gene fromA. usamiiwas optimized forP. pastorisand expressed inP. pastoris. The maximum xylanase activity of optimized (xynB-opt) gene was 33500 U/mL after methanol induction for 144 h in 50 L bioreactor, which was 59% higher than that by wild-type (xynB) gene. To further increase the expression ofxynB-opt, theVitreoscilla hemoglobin(VHb) gene was transformed to the recombinant strain containingxynB-opt. The results showed that recombinant strain harboring thexynB-optandVHb(named X33/xynB-opt-VHb) displayed higher biomass, cell viability, and xylanase activity. The maximum xylanase activity of X33/xynB-opt-VHbin 50 L bioreactor was 45225 U/mL, which was 35% and 115% higher than that by optimized (xynB-opt) gene and wild-type (xynB) gene. Finally, the induction temperature of X33/xynB-opt-VHbwas optimized in 50 L bioreactor. The maximum xylanase activity of X33/xynB-opt-VHbreached 58792 U/mL when the induction temperature was 22°C. The results presented here will greatly contribute to improving the production of recombinant proteins inP. pastoris.

scholarly journals Graph Constraint and Collaborative Representation Classifier Steered Discriminative Projection with Applications for the Early Identification of Cucumber Diseases

Sensors ◽  
2020 ◽  
Vol 20 (4) ◽  
pp. 1217 ◽  
Author(s):  
Yuhua Li ◽  
Fengjie Wang ◽  
Ye Sun ◽  
Yingxu Wang
Keyword(s):  
Early Identification ◽  
Early Stage ◽  
Dimensional Space ◽  
Decision Criterion ◽  
Hyperspectral Data ◽  
Identification Accuracy ◽  
Collaborative Representation ◽  
Efficient Production ◽  
Spectral Curves ◽  
Low Dimensional

Accurate, rapid and non-destructive disease identification in the early stage of infection is essential to ensure the safe and efficient production of greenhouse cucumbers. Nevertheless, the effectiveness of most existing methods relies on the disease already exhibiting obvious symptoms in the middle to late stages of infection. Therefore, this paper presents an early identification method for cucumber diseases based on the techniques of hyperspectral imaging and machine learning, which consists of two procedures. First, reconstruction fidelity terms and graph constraints are constructed based on the decision criterion of the collaborative representation classifier and the desired spatial distribution of spectral curves (391 to 1044 nm) respectively. The former constrains the same-class and different-class reconstruction residuals while the latter constrains the weighted distances between spectral curves. They are further fused to steer the design of an offline algorithm. The algorithm aims to train a linear discriminative projection to transform the original spectral curves into a low dimensional space, where the projected spectral curves of different diseases own better separation trends. Then, the collaborative representation classifier is utilized to achieve online early diagnosis. Five experiments were performed on the hyperspectral data collected in the early infection stage of cucumber anthracnose and Corynespora cassiicola diseases. Experimental results demonstrated that the proposed method was feasible and effective, providing a maximal identification accuracy of 98.2% and an average online identification time of 0.65 ms. The proposed method has a promising future in practical production due to its high diagnostic accuracy and short diagnosis time.

Effect of vitamin D treatment on VDR expression in primary cerebral cortical cells in induced oxidative stress

2020 ◽  
pp. 1-10
Author(s):  
Ebtesam Alsulami ◽  
Majed Alokail ◽  
Amani Alghamedi ◽  
Abir Alamro ◽  
Samina Haq
Keyword(s):  
Oxidative Stress ◽  
Vitamin D ◽  
Vitamin D3 ◽  
Cultured Cells ◽  
Neuroprotective Effect ◽  
Sandwich Elisa ◽  
Stress Protein ◽  
Neuronal Cultures ◽  
Primary Neuronal Cultures ◽  
Peripheral Tissues

BACKGROUND: In addition to calcium and phosphate homeostasis in peripheral tissues; vitamin D performs a neuroprotection role in the nervous system. The neuroprotective actions of vitamin D include: increasing vitamin D receptor (VDR) expression, control glutathione synthesis and nitric oxide synthase activity and induce neurotrophins such as nerve growth factor (NGF). VDR mediates cellular actions, and biological responses of the vitamin D. OBJECTIVE: To study the effect of VDR and NGF expression levels by vitamin D3 treatment in induced oxidative stress in primary cortical neuronal cultures. METHOD: Primary neuronal cultures were set up from the cortex region of neonatal rat’s brain. They were cultured for up to 72 h in the presence of 0.25μg/ml vitamin D3. These cells were exposed to 0.5 mM H2O2 for two hours before collecting cell pellet and medium for biochemical assays. Control and H2O2 treated cells were cultured in the absence of vitamin D3 treatment. Sandwich ELISA was used to study NGF expression. Western blotting and Immunofluorescence of cultured cells were used to estimate the expression of VDR. RESULTS: Vitamin D3 treatment increased more significantly (P <  0.001) NGF levels with and without induced oxidative stress. Protein expression studies confirmed the positive correlation between VDR expression and vitamin D3 treatment after 72 h in culture. Moreover, pre-treating the cells with vitamin D3 before H2O2 exposure significantly increase (P <  0.05) VDR expression in comparison with the cells exposed to H2O2 alone. CONCLUSION: The neuroprotective effect of vitamin D3 against oxidative stress could be through up-regulating VDR and NGF levels.

scholarly journals Functional expression of the lactate permease Jen1p of Saccharomyces cerevisiae in Pichia pastoris

2003 ◽  
Vol 376 (3) ◽  
pp. 781-787 ◽  
Author(s):  
Isabel SOARES-SILVA ◽  
Dorit SCHULLER ◽  
Raquel P. ANDRADE ◽  
Fátima BALTAZAR ◽  
Fernanda CÁSSIO ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Acetic Acid ◽  
Lactic Acid ◽  
Pichia Pastoris ◽  
Plasma Membranes ◽  
Membrane Vesicle ◽  
Dry Weight ◽  
Functional Expression ◽  
Acid Uptake ◽  
Methanol Induction

In Saccharomyces cerevisiae the activity for the lactate–proton symporter is dependent on JEN1 gene expression. Pichia pastoris was transformed with an integrative plasmid containing the JEN1 gene. After 24 h of methanol induction, Northern and Western blotting analyses indicated the expression of JEN1 in the transformants. Lactate permease activity was obtained in P. pastoris cells with a Vmax of 2.1 nmol·s−1·mg of dry weight−1. Reconstitution of the lactate permease activity was achieved by fusing plasma membranes of P. pastoris methanol-induced cells with Escherichia coli liposomes containing cytochrome c oxidase, as proton-motive force. These assays in reconstituted heterologous P. pastoris membrane vesicles demonstrate that S. cerevisiae Jen1p is a functional lactate transporter. Moreover, a S. cerevisiae strain deleted in the JEN1 gene was transformed with a centromeric plasmid containing JEN1 under the control of the glyceraldehyde-3-phosphate dehydrogenase constitutive promotor. Constitutive JEN1 expression and lactic acid uptake were observed in cells grown on either glucose and/or acetic acid. The highest Vmax (0.84 nmol·s−1·mg of dry weight−1) was obtained in acetic acid-grown cells. Thus overexpression of the S. cerevisiae JEN1 gene in both S. cerevisiae and P. pastoris cells resulted in increased activity of lactate transport when compared with the data previously reported in lactic acid-grown cells of native S. cerevisiae strains. Jen1p is the only S. cerevisiae secondary porter characterized so far by heterologous expression in P. pastoris at both the cell and the membrane-vesicle levels.

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