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Order Blubervirales: Surface Protein
Published in Paul Pumpens, Peter Pushko, Philippe Le Mercier, Virus-Like Particles, 2022
Paul Pumpens, Peter Pushko, Philippe Le Mercier
Second, yeast Pichia pastoris was used for another set of hepatitis B vaccines. They were Cuban SHBs-based vaccine Heberbiovac HB elaborated by the Centro de Ingenieria Genetica Y Biotecnologia and produced by the Heberbiotec SA (Cuba) company (Galbán García et al. 1992) and its analogue, which was produced in India under the trademark Enivac HB (Kaur and Mani 2000; Estévez et al. 2007). A low-cost P. pastoris-produced vaccine Shanvac B was registered in India (Abraham et al. 1999).
Recombinant Antibodies
Published in Siegfried Matzku, Rolf A. Stahel, Antibodies in Diagnosis and Therapy, 2019
Melvyn Little, Sergey M. Kipriyanov
The methylotrophic yeast Pichia pastoris has been shown to be suitable for the high-level expression of various heterologous proteins, either intracellular or secreted into the culture supernatant (see review by Cregg et al., 1993). Recently, Ridder et al. (1995) demonstrated the applicability of the Pichia expression system for the secretion of antibody fragments. A rabbit scFv against the human leukemia inhibitory factor (hLIF) was expressed in Pichia cells using the expression vector pPIC9 that provides the α-mating factor signal sequence for secretion and the HIS4 gene for selection of the recombinant yeast clones. The yield was about 100 mg per liter of shake flask culture.
Potential of Antibody Therapy for Respiratory Virus Infections
Published in Sunit K. Singh, Human Respiratory Viral Infections, 2014
Tze-Minn Mak, Ruisi Hazel Lin, Yee-Joo Tan
Most yeast display technologies are based on Saccharomyces cerevisiae, however. Pichia pastoris has been introduced as an alternative species as it does not revert to anaerobic metabolism under oxygen deprivation conditions, and thus does not produce toxic metabolites. It is also easier to genetically modify and express, and is stable in both haploid and diploid forms [75]. However, the posttranslational glycosylation in P. pastoris differs greatly from humans and could result in immunogenicity if these mAbs are used for human therapy. Methods to fine-tune glycosylation of antibodies in these systems, such as the removal of genes encoding certain mannose sugars in P. pastoris pathways [76], are currently being studied.
Genetically modified Pichia pastoris, a powerful resistant factory for gold and palladium bioleaching and nanostructure heavy metal biosynthesis
Published in Artificial Cells, Nanomedicine, and Biotechnology, 2020
Fatemeh Elahian, Razieh Heidari, Vahid Reza Charghan, Elham Asadbeik, Seyed Abbas Mirzaei
The previous study represented that engineered Pichia pastoris is an efficient option for the silver and selenium uptake and biotransformation. Cytochrome-b5 reductase arms Pichia to be a metal-resistant cell and to survive in high concentrations of the heavy metal ions. NADH-dependent reductase enzyme mediates the electron transport from NADH to a single FAD group. Then, the prosthetic FAD domain directly catalyzes the stoichiometric transfer of reducing equivalents, electrons, to the small heavy metal ion partners. Such engineered yeast usage for nanoparticle productions gain several advantages over physicochemical methods, immobilized enzyme bioreactors and non-transformed factory cells due ease of use, automation and the cell manipulation, inexpensive growth requirements and investment, simple scaling-up, high biomass yield, time-/cost-effectiveness, cofactors' needless and eco-friendliness. On the other hand, biosynthetic routes could provide good control over the recombinant enzyme production in the cell and consequently size distribution of nanoparticles than some of the physicochemical methods. Such properties introduced the developed microorganism as an excellent tool in nanotechnology science [16,21–24].
Nebulized delivery of a broadly neutralizing SARS-CoV-2 RBD-specific nanobody prevents clinical, virological, and pathological disease in a Syrian hamster model of COVID-19
Published in mAbs, 2022
Thomas J. Esparza, Yaozong Chen, Negin P. Martin, Helle Bielefeldt-Ohmann, Richard A. Bowen, William D. Tolbert, Marzena Pazgier, David L. Brody
Using the previously characterized11 strain of X-33 Pichia pastoris expressing NIH-CoVnb-112, fermentation was performed to produce sufficient material for in vivo studies and reduce the risk of endotoxin contamination. Fermentation was performed by the University of Georgia Bioexpression and Fermentation Facility (UGA BFF, Athens, GA) and performed as briefly follows: A 5–10% inoculum of the expression clone in buffered glycerol complex medium (BMGY) was grown for 24 hours at 30°C. The overnight culture (300 mL) was used to inoculate a 4-L DCI Tryton bioreactor containing 2.7 L basal salts medium (BSM) for a 75% working volume. Post-batch growth was fed with 50% v/v glycerol until the wet cell weight reached 200 grams/L at which point feed was switched to methanol containing 12 mL/L Pichia trace metal salts for induction of expression. Final methanol concentration was maintained in the fermenter at 1 gram/L for 72 hours. Following harvest and clarification, the supernatant volume was reduced 10-fold and exchanged to buffer (20 mMTris, pH 7.4, 0.5 M NaCl) using tangential flow filtration. NIH-CoVnb-112 was affinity purified using a 5 mL HiTrap HP column (Cytiva 17115201) charged with nickel. The sample was eluted using a linear gradient of imidazole to 0.5 M. The eluted nanobody was buffer exchanged to 0.9% normal saline and the final concentration determined based on ProtParam44 mass extinction coefficient estimate (1 mg/mL solution will have an A280 absorbance value of 2.0310) for a yield of 1.1 g. The purity (95.5%) was determined using SDS-PAGE. Endotoxin level was measured using the LAL Chromogenic Endotoxin Quantitation Kit (Cat# 88282, Thermo Scientific) to verify a concentration of less than 0.1 endotoxin unit/mL (EU/mL) of purified nanobody.
TNB-738, a biparatopic antibody, boosts intracellular NAD+ by inhibiting CD38 ecto-enzyme activity
Published in mAbs, 2022
Harshad S. Ugamraj, Kevin Dang, Laure-Hélène Ouisse, Benjamin Buelow, Eduardo N. Chini, Giulia Castello, James Allison, Starlynn C Clarke, Laura M. Davison, Roland Buelow, Rong Deng, Suhasini Iyer, Ute Schellenberger, Sankar N. Manika, Shipra Bijpuria, Astrid Musnier, Anne Poupon, Maria Cristina Cuturi, Wim van Schooten, Pranjali Dalvi
CD38 (UniProt: P28907) was cloned in Pichia pastoris-based expression vector to yield the expression of 45–300 amino acid of CD38, which includes mismatches at 5 positions in the CD38 sequence, in fusion with His-tag at its C-terminus. The fusion protein was expressed by transforming in Pichia pastoris strain BICC 9450. The protein was purified from the supernatant by affinity column chromatography. To obtain a homogenous form of the protein, it was further purified by gel filtration chromatography using Superdex-75 column. The protein was eluted in 20 mM HEPES buffer, pH 7.2 with 50 mM NaCl.