- PRODUCTION AND BIOTECHNOLOGICAL APPLICATIONS OF RECOMBINANT PROTEINS BY METHYLOTROPHIC YEAST: PAST, PRESENT AND FUTURE PERSPECTIVES

2014 ◽  
pp. 222-253
Keyword(s):  
Recombinant Proteins ◽  
Methylotrophic Yeast ◽  
Biotechnological Applications ◽  
Future Perspectives

scholarly journals Ktr1p is an α-1,2-mannosyltransferase of Saccharomyces cerevisiae. Comparison of the enzymic properties of soluble recombinant Ktr1p and Kre2p/Mnt1p produced in Pichia pastoris

1997 ◽  
Vol 321 (2) ◽  
pp. 289-295 ◽  
Author(s):  
Pedro A. ROMERO ◽  
Marc LUSSIER ◽  
Anne-Marie SDICU ◽  
Howard BUSSEY ◽  
Annette HERSCOVICS
Keyword(s):  
Pichia Pastoris ◽  
Recombinant Proteins ◽  
Methylotrophic Yeast ◽  
Secreted Proteins ◽  
Yeast Genome ◽  
Amino Acid Similarity ◽  
Substrate Specificities ◽  
Yeast Pichia Pastoris ◽  
Methylotrophic Yeast Pichia Pastoris ◽  
Absolute Requirement

The yeast genome contains a KRE2/MNT1 family of nine related genes with amino acid similarity to the α1,2-mannosyltransferase Kre2p/Mnt1p, the only member of this family whose enzymic properties have been studied. In this study, the enzymic properties of Ktr1p, another member of this family, were studied and compared to those of Kre2p/Mnt1p. Recombinant soluble forms of Kre2p/Mnt1p and Ktr1p lacking their N-terminal regions were expressed as secreted proteins from the methylotrophic yeast Pichia pastoris. After induction with methanol, the medium contained approx. 40 and 400 mg/l of soluble recombinant Kre2p/Mnt1p and Ktr1p respectively. Both recombinant proteins were shown to exhibit α1,2-mannosyltransferase activity. The enzymes have an absolute requirement for Mn2+ and a similar Km for mannose (280Ő350 mM), methyl-α-mannoside (60Ő90 mM) and GDP-mannose (50Ő90 ƁM), but the Vmax was approx. 10 times higher for Kre2p/Mnt1p than for Ktr1p. The enzymes have similar substrate specificities and utilize mannose, methyl-α-mannoside, α-1,2-mannobiose and methyl-α-1,2-mannobiose, as well as Man15Ő30GlcNAc, derived from mnn2 mutant glycoproteins, as substrates. The enzymes do not utilize α-1,6-mannobiose, α-1,6-mannotriose, α-1,6-mannotetraose, mammalian Man9GlcNAc or yeast Man9Ő10GlcNAc. These results indicate that Kre2p/Mnt1p and Ktr1p are capable of participating in both N-glycan and O-glycan biosynthesis.

Aminomutases: mechanistic diversity, biotechnological applications and future perspectives

2011 ◽  
Vol 29 (7) ◽  
pp. 352-362 ◽  
Author(s):  
Bian Wu ◽  
Wiktor Szymański ◽  
Matthew M. Heberling ◽  
Ben L. Feringa ◽  
Dick B. Janssen
Keyword(s):  
Biotechnological Applications ◽  
Future Perspectives

scholarly journals The Expanding Role of Vesicles Containing Aquaporins

Cells ◽  
2018 ◽  
Vol 7 (10) ◽  
pp. 179 ◽  
Author(s):  
M Martinez-Ballesta ◽  
Paula Garcia-Ibañez ◽  
Lucía Yepes-Molina ◽  
Juan Rios ◽  
Micaela Carvajal
Keyword(s):  
Environmental Changes ◽  
Cell Communication ◽  
Membrane Vesicles ◽  
Living Cells ◽  
Biotechnological Applications ◽  
Future Perspectives ◽  
Physiological Processes ◽  
Communication Processes ◽  
Other Substances

In animals and plants, membrane vesicles containing proteins have been defined as key for biological systems involving different processes such as trafficking or intercellular communication. Docking and fusion of vesicles to the plasma membrane occur in living cells in response to different stimuli, such as environmental changes or hormones, and therefore play an important role in cell homeostasis as vehicles for certain proteins or other substances. Because aquaporins enhance the water permeability of membranes, their role as proteins immersed in vesicles formed of natural membranes is a recent topic of study. They regulate numerous physiological processes and could hence serve new biotechnological purposes. Thus, in this review, we have explored the physiological implications of the trafficking of aquaporins, the mechanisms that control their transit, and the proteins that coregulate the migration. In addition, the importance of exosomes containing aquaporins in the cell-to-cell communication processes in animals and plants have been analyzed, together with their potential uses in biomedicine or biotechnology. The properties of aquaporins make them suitable for use as biomarkers of different aquaporin-related diseases when they are included in exosomes. Finally, the fact that these proteins could be immersed in biomimetic membranes opens future perspectives for new biotechnological applications.

scholarly journals Recent Advancements and Future Perspectives of Microalgae-Derived Pharmaceuticals

Marine Drugs ◽  
2021 ◽  
Vol 19 (12) ◽  
pp. 703
Author(s):  
Donghua Xia ◽  
Wen Qiu ◽  
Xianxian Wang ◽  
Junying Liu
Keyword(s):  
Recombinant Proteins ◽  
Large Scale ◽  
Low Cost ◽  
Commercial Production ◽  
Future Perspectives ◽  
Cell Factories ◽  
Microalgal Biotechnology ◽  
Medicinal Properties ◽  
Solar Powered ◽  
Pathogenic Contamination

Microalgal cells serve as solar-powered factories that produce pharmaceuticals, recombinant proteins (vaccines and drugs), and valuable natural byproducts that possess medicinal properties. The main advantages of microalgae as cell factories can be summarized as follows: they are fueled by photosynthesis, are carbon dioxide-neutral, have rapid growth rates, are robust, have low-cost cultivation, are easily scalable, pose no risk of human pathogenic contamination, and their valuable natural byproducts can be further processed. Despite their potential, there are many technical hurdles that need to be overcome before the commercial production of microalgal pharmaceuticals, and extensive studies regarding their impact on human health must still be conducted and the results evaluated. Clearly, much work remains to be done before microalgae can be used in the large-scale commercial production of pharmaceuticals. This review focuses on recent advancements in microalgal biotechnology and its future perspectives.

scholarly journals Fhl1p protein, a positive transcription factor in Pichia pastoris, enhances the expression of recombinant proteins

2019 ◽  
Vol 18 (1) ◽  
Author(s):  
Xueyun Zheng ◽  
Yimin Zhang ◽  
Xinying Zhang ◽  
Cheng Li ◽  
Xiaoxiao Liu ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Pichia Pastoris ◽  
Recombinant Proteins ◽  
Recombinant Protein Expression ◽  
Large Subunit ◽  
Protein A ◽  
Methylotrophic Yeast ◽  
Regulation Mechanism ◽  
Heterologous Proteins ◽  
Foreign Proteins

Abstract Background The methylotrophic yeast Pichia pastoris is well-known for the production of a broad spectrum of functional types of heterologous proteins including enzymes, antigens, engineered antibody fragments, and next gen protein scaffolds and many transcription factors are utilized to address the burden caused by the high expression of heterologous proteins. In this article, a novel P. pastoris transcription factor currently annotated as Fhl1p, an activator of ribosome biosynthesis processing, was investigated for promoting the expression of the recombinant proteins. Results The function of Fhl1p of P. pastoris for improving the expression of recombinant proteins was verified in strains expressing phytase, pectinase and mRFP, showing that the productivity was increased by 20–35%. RNA-Seq was used to study the Fhl1p regulation mechanism in detail, confirming Fhl1p involved in the regulation of rRNA processing genes, ribosomal small/large subunit biogenesis genes, Golgi vesicle transport genes, etc., which contributed to boosting the expression of foreign proteins. The overexpressed Fhl1p strain exhibited increases in the polysome and monosome levels, showing improved translation activities. Conclusion This study illustrated that the transcription factor Fhl1p could effectively enhance recombinant protein expression in P. pastoris. Furthermore, we provided the evidence that overexpressed Fhl1p was related to more active translation state.

scholarly journals Evaluación del tiempo de inducción y la concentración de metanol en la expresión de L-asparaginasa II de Saccharomyces cerevisiae usando Pichia pastoris (Muts)

2018 ◽  
pp. 131-134
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Pichia Pastoris ◽  
Recombinant Proteins ◽  
Induction Time ◽  
Lymphoblastic Leukemia ◽  
Methylotrophic Yeast ◽  
Culture Conditions ◽  
Methylotrophic Yeast Pichia Pastoris ◽  
Low Concentrations ◽  
Eukaryotic Organisms

Evaluación del tiempo de inducción y la concentración de metanol en la expresión de L-asparaginasa II de Saccharomyces cerevisiae usando Pichia pastoris (Muts) Omar Pillaca-Pullo, Karin Torres, Marcela Pimenta, Adalberto Pessoa-Jr & Michele Vitolo Facultad de Ciencias Farmacéuticas – Universidad de São Paulo (Brasil), 0550-8000 DOI: https://doi.org/10.33017/RevECIPeru2015.0020/ Resumen La levadura metilotrófica Pichia pastoris es ampliamente usada como un sistema eucariota para expresar proteínas recombinantes. Más de 500 proteínas recombinantes fueron expresas por P. pastoris con niveles de expresión que alcanzan hasta el 80% de proteínas totales secretadas y hasta 30% de proteínas totales de la célula. Existen tres fenotipos de P. pastoris clasificados de acuerdo con su capacidad de metabolizar metanol, el fenotipo MutS crece lentamente en medios con metanol por lo que generalmente se usan bajas concentraciones de metanol y tiempo de inducción prolongados. Por esta razón, el control de las condiciones de cultura como la concentración del inductor y el tiempo de inducción son factores importantes tanto para el crecimiento de la levadura como para producción de la proteína ya que este sistema es controlado por el promotor AOX inducido con metanol. Por otro lado, L-asparaginase (EC. 3.5.1.1) es un importante biofármaco usado en el tratamiento de leucemia linfoblástica aguda (ALL), la enzima comúnmente utilizada en la terapéutica es procedente de bacterias, estas han demostrado buena actividad, pero causan muchas reacciones inmunológicas severas en los pacientes tratados. La búsqueda de L-asparaginasa procedente y expresada en organismos eucariotas se abre como una posibilidad para reducir las reacciones adversas. En este estudio fueron evaluados el tiempo de inducción (24 - 120 horas) y la concentración de inductor (0.25, 0.5 y 1.0%). Los datos mostraron que la condición de mayor expresión de L-asparaginasa II  de Saccharomyces cerevisiae después de 48 horas de inducción con 1,0% de metanol (~ 25 U.g-1). Finalmente se recomienda evaluar dicha producción en biorreactor donde se lleve un control adecuado de otras variables importantes como el pH del cultivo y la concentración de oxígeno en el medio. Descriptores: Pichia pastoris, Saccharomyces cerevisiae, L-asparaginase, Metanol.  Abstract The methylotrophic yeast Pichia pastoris is widely used as a eukaryotic system for expressing recombinant proteins. Over 500 recombinant proteins were expressed in P. pastoris with expression levels reaching up to 80% of total secreted proteins and up to 30% total cell proteins. There are three phenotypes of P. pastoris classified according to their ability to metabolize methanol, phenotype MutS grows slowly on media containing methanol at generally low concentrations of methanol and longer induction time are used. Therefore, the control of culture conditions such as concentration of the inducer and the induction time are important factors for both yeast growth and for production of the protein since this system is controlled by the AOX promoter induced with methanol. Furthermore, L-asparaginase (EC. 3.5.1.1) is an important biopharmaceutical used to treat acute lymphoblastic leukemia (ALL), the enzyme commonly used in the therapy is from bacteria, these have shown good activity but cause many severe immune reactions in patients. The search for L-asparaginase derived and expressed in eukaryotic organisms opens a possibility to reduce adverse reactions. In this study they were evaluated the induction time (24-120 hours) and inducer concentration (0.25, 0.5 and 1.0% v/v). The data showed that the condition of increased expression of L-asparaginase II from Saccharomyces cerevisiae after 48 hours’ induction with 1.0% methanol (~ 25 U.g-1). Finally, it is recommended to evaluate this production in bioreactor where adequate control of other important variables such as pH of the culture and the concentration of oxygen in the medium is carried. Keywords: Pichia pastoris, Saccharomyces cerevisiae, L-asparaginase, Metanol.

scholarly journals The Mut+strain ofKomagataella phaffii(Pichia pastoris) expresses PAOX15 and 10 times faster than Mutsand Mut−strains: Evidence that formaldehyde or/and formate are true inducers of AOX

2019 ◽  
Author(s):  
Anamika Singh ◽  
Atul Narang
Keyword(s):  
Protein Synthesis ◽  
Recombinant Protein ◽  
Recombinant Proteins ◽  
Methylotrophic Yeast ◽  
Inducible Promoter ◽  
Wild Type ◽  
Methanol Metabolism ◽  
In The Wild ◽  
Host Strains ◽  
Better Than

AbstractThe methylotrophic yeastKomagataella phaffiiis among the most popular hosts for recombinant protein synthesis. Most recombinant proteins were expressed in the wild-type Mut+host strain from the methanol-inducible promoter PAOX1. Since methanol metabolism has undesirable consequences, two additional host strains, Muts(AOX1-) and Mut−(AOX1-AOX2-), were introduced which consume less methanol and reportedly also express recombinant protein better than Mut+. Both results follow from a simple model based on two widespread assumptions, namely methanol is transported by diffusion and the sole inducer of PAOX1. To test this model, we studied14C-methanol uptake in the Mut−strain and β-galactosidase expression in all three strains. We confirmed that methanol is transported by diffusion, but in contrast to the literature, Mut+expressed β-galactosidase 5- and 10-fold faster than Mutsand Mut−. These results imply that methanol is not the sole inducer of PAOX1— metabolites downstream of methanol also induce PAOX1. We find that formate or/and formaldehyde are probably true inducers since both induce PAOX1expression in Mut−which cannot synthesize intracellular methanol from formate or formaldehyde. Formate offers a promising substitute for methanol since it does not appear to suffer from the deficiencies that afflict methanol.

scholarly journals Plant Serine Protease Inhibitors: Biotechnology Application in Agriculture and Molecular Farming

2019 ◽  
Vol 20 (6) ◽  
pp. 1345 ◽  
Author(s):  
Marina Clemente ◽  
Mariana Corigliano ◽  
Sebastián Pariani ◽  
Edwin Sánchez-López ◽  
Valeria Sander ◽  
...  
Keyword(s):  
Serine Protease ◽  
Protease Inhibitors ◽  
Recombinant Proteins ◽  
Molecular Farming ◽  
Physiological Role ◽  
Main Function ◽  
Biotechnological Applications ◽  
Serine Protease Inhibitors ◽  
Living Organisms ◽  
Phytopathogenic Microorganisms

The serine protease inhibitors (SPIs) are widely distributed in living organisms like bacteria, fungi, plants, and humans. The main function of SPIs as protease enzymes is to regulate the proteolytic activity. In plants, most of the studies of SPIs have been focused on their physiological role. The initial studies carried out in plants showed that SPIs participate in the regulation of endogenous proteolytic processes, as the regulation of proteases in seeds. Besides, it was observed that SPIs also participate in the regulation of cell death during plant development and senescence. On the other hand, plant SPIs have an important role in plant defense against pests and phytopathogenic microorganisms. In the last 20 years, several transgenic plants over-expressing SPIs have been produced and tested in order to achieve the increase of the resistance against pathogenic insects. Finally, in molecular farming, SPIs have been employed to minimize the proteolysis of recombinant proteins expressed in plants. The present review discusses the potential biotechnological applications of plant SPIs in the agriculture field.

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