scholarly journals Magnaporthe oryzae as an expression host for the production of the unspecific peroxygenase AaeUPO from the basidiomycete Agrocybe aegerita

2021 ◽  
Author(s):  
Stefan Jacob ◽  
Sebastian Bormann ◽  
Michael Becker ◽  
Luis Antelo ◽  
Dirk Holtmann ◽  
...  
Keyword(s):  
Magnaporthe Oryzae ◽  
Posttranslational Modifications ◽  
Enzyme Assay ◽  
Expression System ◽  
Human Organism ◽  
Heterologous Production ◽  
Agrocybe Aegerita ◽  
Filamentous Ascomycete ◽  
Expression Host ◽  
Alternative Expression

The filamentous fungus Magnaporthe oryzae has the potential to be developed as an alternative platform organism for the heterologous production of industrially important enzymes. M. oryzae is easy to handle, fast-growing and unlike yeast, posttranslational modifications like N-glycosylations are similar to the human organism. Here, we established M. oryzae as a host for the expression of the unspecific peroxygenase from the basidiomycete Agrocybe aegerita (AaeUPO). UPOs are attractive biocatalysts for selective oxyfunctionalization of non-activated carbon-hydrogen bonds. To improve and simplify the isolation of AaeUPO in M. oryzae, we fused a Magnaporthe signal peptide for protein secretion and set it under control of the strong EF1-promotor. The success of the heterologous production of full-length AaeUPO in M. oryzae and the secretion of the functional enzyme was confirmed by a peroxygenase-specific enzyme assay. These results offer the possibility to establish the filamentous ascomycete M. oryzae as a broad applicable alternative expression system. This is in particular valid for proteins that cannot or not in sufficient yields produced in established systems.

scholarly journals Magnaporthe oryzae as an expression host for the production of the unspecific peroxygenase Aae UPO from the basidiomycete Agrocybe aegerita

2021 ◽  
Vol 10 (6) ◽  
Author(s):  
Stefan Jacob ◽  
Sebastian Bormann ◽  
Michael Becker ◽  
Luis Antelo ◽  
Dirk Holtmann ◽  
...  
Keyword(s):  
Magnaporthe Oryzae ◽  
Agrocybe Aegerita ◽  
Expression Host

scholarly journals A Newly Designed EGFP-2A Peptide Monocistronic Baculoviral Vector for Concatenating the Expression of Recombinant Proteins in Insect Cells

Processes ◽  
2019 ◽  
Vol 7 (5) ◽  
pp. 291 ◽  
Author(s):  
Chih-Yu Wu ◽  
Chao-Wei Huang ◽  
Yu-Shin Nai ◽  
Pei-Yu Chu ◽  
Chung-Hsiung Wang ◽  
...  
Keyword(s):  
Recombinant Proteins ◽  
Posttranslational Modifications ◽  
Mammalian Cells ◽  
Insect Cells ◽  
Fluorescent Protein ◽  
Expression System ◽  
Recombinant Baculovirus ◽  
New Combination ◽  
Vector System ◽  
Baculovirus Expression Vector

Recombinant proteins produced by the baculovirus expression vector system (BVES) have been widely applied in the agricultural and medical fields. However, the procedure for protein expression is inefficient and needs to be improved. Herein, we propose a simple construct that incorporates a selectable marker (enhanced green fluorescent protein, EGFP) and a picorna viral-derived “self-cleaving” 2A-like peptide to separate the EGFP and target proteins in a monocistronic baculovirus vector to facilitate isolation of the recombinant baculovirus in the BVES. In this study, porcine adiponectin (ADN), a secreted, multimeric protein with insulin-sensitizing properties, was used to demonstrate its utility in our EGFP-2A-based expression system. EGFP and ADN were simultaneously expressed by a recombinant alphabaculovirus. Co-expression of EGFP facilitates the manipulation of the following processes, such as determining expression kinetics and harvesting ADN. The results showed that the 2A “self-cleaving” process does not interfere with EGFP activity or with signal peptide removal and the secretion of recombinant ADN. Posttranslational modifications, including glycosylation, of the recombinant ADN occurred in insect cells, and the formation of various multimers was further verified. Most importantly, the insect-produced ADN showed a similar bioactivity to that of mammalian cells. This concept provides a practical and economic approach that utilizes a new combination of alphabaculovirus/insect cell expression systems for future applications.

Production of Brucella melitensis Omp16 protein fused to the human interleukin 2 in Lactococcus lactis MG1363 toward developing a Lactococcus-based vaccine against brucellosis

2020 ◽  
Vol 66 (1) ◽  
pp. 39-45
Author(s):  
Marzieh Rezaei ◽  
Mohammad Rabbani Khorasgani ◽  
Sayyed Hamid Zarkesh Esfahani ◽  
Rahman Emamzadeh ◽  
Hamid Abtahi
Keyword(s):  
Fusion Protein ◽  
Lactococcus Lactis ◽  
Fusion Gene ◽  
Brucella Melitensis ◽  
Expression System ◽  
Interleukin 2 ◽  
Cell Factory ◽  
Promising Alternative ◽  
Alternative Expression ◽  
Restriction Sites

The use of the food-grade bacterium Lactococcus lactis as a new cell factory is a promising alternative expression system for producing a desired protein. The Omp16-IL2 fusion protein antigen was cloned, expressed, and purified in this study. The Omp16-IL2 fusion gene was designed and cloned in pGH plasmid with appropriate restriction sites and subcloned in pAMJ2008 expression vector digested with the same enzymes. The purified recombinant constructed pAMJ-rOmp-IL2 was introduced into L. lactis subsp. cremoris MG1363 by electrotransformation. Finally, the expression and purification of Omp16-IL2 fusion protein was investigated. This study reports the construction of a recombinant L. lactis expressing the Omp16-IL2 fusion protein as an oral Lactococcus-based vaccine, as compared with commonly used live attenuated vaccines, for future studies against brucellosis.

scholarly journals A newly isolated yeast as an expression host for recombinant lipase

2015 ◽  
Vol 20 (2) ◽  
Author(s):  
Siti Nurbaya Oslan ◽  
Abu Bakar Salleh ◽  
Raja Noor Zaliha Raja Abd Rahman ◽  
Thean Chor Leow ◽  
Hafizah Sukamat ◽  
...  
Keyword(s):  
Specific Activity ◽  
Alcohol Oxidase ◽  
Yeast Genome ◽  
Optimum Temperature ◽  
Pichia Guilliermondii ◽  
Methanol Induction ◽  
Expression Host ◽  
Alternative Expression ◽  
Wide Ph Range ◽  
Ph Range

AbstractPichia guilliermondii strain SO isolated from spoiled orange was developed for use as an alternative expression host by using Pichia pastoris as the model of the experiment. This is the first study to report on the capability of P. guilliermondii SO as a host to express thermostable T1 lipase from Geobacillus zalihae. Alcohol oxidase and formaldehyde dehydrogenase promoters were present in the yeast genome. Interestingly, the recombinant yeast [SO/pPICZαB/T1-2 (SO2)] took only 30 h to reach optimal production with minimal methanol induction [1.5% (v/v)] in YPTM medium, as compared to P. pastoris, which took longer to reach its optimal condition. The purification yield of the His-tagged fusion lipase was 68.58%, with specific activity of 194.58 U/mg. The optimum temperature was 65°C at pH 9 in glycine-NaOH buffer, and it was stable up to 70°C in a wide pH range from pH 5 to 12. In conclusion, a newly isolated yeast from spoiled orange has been proven suitable for use as an expression host.

scholarly journals Rhinella marina oocytes: a suitable alternative expression system for functional characterization of aquaglyceroporins

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Vania Rojas ◽  
Yulexi Y. Ortiz ◽  
Sheridan Rodríguez ◽  
Vladimir Araque ◽  
Alexis Rodríguez-Acosta ◽  
...  
Keyword(s):  
Expression System ◽  
Functional Characterization ◽  
Suitable Alternative ◽  
Rhinella Marina ◽  
Alternative Expression

scholarly journals Holotoxin Activity of Botulinum Neurotoxin Subtype A4 Originating from a Nontoxigenic Clostridium botulinum Expression System

2014 ◽  
Vol 80 (23) ◽  
pp. 7415-7422 ◽  
Author(s):  
Marite Bradshaw ◽  
William H. Tepp ◽  
Regina C. M. Whitemarsh ◽  
Sabine Pellett ◽  
Eric A. Johnson
Keyword(s):  
Clostridium Botulinum ◽  
Botulinum Neurotoxin ◽  
Expression System ◽  
Single Amino Acid ◽  
Content Type ◽  
Expression Host ◽  
Toxin Complex ◽  
Human Neuronal Cells

ABSTRACTClostridium botulinumsubtype A4 neurotoxin (BoNT/A4) is naturally expressed in the dual-toxin-producingC. botulinumstrain 657Ba at 100× lower titers than BoNT/B. In this study, we describe purification of recombinant BoNT/A4 (rBoNT/A4) expressed in a nonsporulating and nontoxigenicC. botulinumexpression host strain. The rBoNT/A4 copurified with nontoxic toxin complex components provided intransby the expression host and was proteolytically cleaved to the active dichain form. Activity of the recombinant BoNT/A4 in mice and in human neuronal cells was about 1,000-fold lower than that of BoNT/A1, and the recombinant BoNT/A4 was effectively neutralized by botulism heptavalent antitoxin. A previous report using recombinant truncated BoNT/A4 light chain (LC) expressed inEscherichia colihas indicated reduced stability and activity of BoNT/A4 LC compared to BoNT/A1 LC, which was surmounted by introduction of a single-amino-acid substitution, I264R. In order to determine whether this mutation would also affect the holotoxin activity of BoNT/A4, a recombinant full-length BoNT/A4 carrying this mutation as well as a second mutation predicted to increase solubility (L260F) was produced in the clostridial expression system. Comparative analyses of thein vitro, cellular, andin vivoactivities of rBoNT/A4 and rBoNT/A4-L260F I264R showed 1,000-fold-lower activity than BoNT/A1 in both the mutated and nonmutated BoNT/A4. This indicates that these mutations do not alter the activity of BoNT/A4 holotoxin. In summary, a recombinant BoNT from a dual-toxin-producing strain was expressed and purified in an endogenous clostridial expression system, allowing analysis of this toxin.

Isotope Depletion Mass Spectrometry (ID-MS) for Enhanced Top-Down Protein Fragmentation

2018 ◽  
Author(s):  
Kelly J. Gallagher ◽  
Michael Palasser ◽  
Sam Hughes ◽  
C. Logan Mackay ◽  
David P. A. Kilgour ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Molecular Mass ◽  
Posttranslational Modifications ◽  
Recombinant Expression ◽  
Expression System ◽  
Single Amino Acid ◽  
Sequence Coverage ◽  
Top Down ◽  
Fragment Ions ◽  
Signal Overlap

<div>Top-down mass spectrometry has become an important technique for the identification of proteins and characterisation of chemical and posttranslational modifications. However, as the molecular mass of proteins increases intact mass determination and top-down fragmentation efficiency become more challenging due to the partitioning of the mass spectral signal into many isotopic peaks. In large proteins, this results in reduced sensitivity and increased spectral complexity and signal overlap. This phenomenon is a consequence of the natural isotopic heterogeneity of the elements which comprise proteins (notably 13C). Here we present a bacterial recombinant expression system for the production of proteins depleted in 13C and 15N and use this strategy to prepare a range of isotopically depleted proteins. High resolution MS of isotope depleted proteins reveal dramatically reduced isotope distributions, which results in increases in sensitivity and deceased spectral complexity. We demonstrate that the monoisotopic signal is observed in mass spectra of proteins up to ~50 kDa. This allows confident assignment of accurate molecular mass, and facile detection of low mass modifications (such as deamidation). We outline the benefits of this isotope depletion strategy for top-down fragmentation. The reduced spectral complexity alleviates problems of signal overlap; the presence of monoisotopic signals allow more accurate assignment of fragment ions; and the dramatic increase in single-to-noise ratio (up to 7-fold increases) permits vastly reduced data acquisition times. Together, these compounding benefits allow the assignment of ca. 3-fold more fragment ions than analysis of proteins with natural isotopic abundances. Thus, more comprehensive sequence coverage can be achieved; we demonstrate near single amino-acid resolution of the 29 kDa protein carbonic anhydrase from a single top-down MS experiment. Finally, we demonstrate that the ID-MS strategy allows far greater sequence coverage to be obtained in time limited top-down data acquisitions – highlighting potential advantages for top-down LC-MS/MS workflows and top-down proteomics. </div><div><br></div>

Acetolactate synthase activity and chlorsulfuron sensitivity of gamma-irradiated lentil (Lens culinaris Medik.) cultivars

2003 ◽  
Vol 140 (1) ◽  
pp. 83-91 ◽  
Author(s):  
H. I. MALKAWI ◽  
N. A. AL-QURAAN ◽  
W. M. OWAIS
Keyword(s):  
Lens Culinaris ◽  
Enzyme Assay ◽  
Expression System ◽  
Acetolactate Synthase ◽  
Plant Tolerance ◽  
High Concentration ◽  
Biochemical Basis ◽  
Tolerant Plants ◽  
Herbicide Binding ◽  
Gamma Irradiated

Lentil (Lens culinaris Medik.) cultivars (Jordan 1 and Jordan 2) were sensitized to chlorsulfuron herbicide. Both cultivars were subjected to three doses of gamma-irradiation (90, 100 and 110 Gray) to develop plants tolerant/resistant to this herbicide. The herbicide-tolerant plants as well as the sensitized plants were subjected to acetolactate synthase (ALS) enzyme assay to reveal the biochemical basis of plant tolerance. The results indicated that as the radiation dose increased, the tolerance to the herbicide application decreased in both lentil cultivars. Cultivar Jordan 1 showed sensitivity to chlorsulfuron herbicide at all treatments compared with that of Jordan 2. ALS enzyme activity in the two lentil cultivars was inhibited by chlorsulfuron. The irradiated plants (90 Gray) in both treated seeds plants (M1) and seeds of tolerant plants (M2) showed a lower level of inhibition to high concentration (250 μg/l) of chlorsulfuron herbicide. The results suggested an alteration of the expression system of ALS gene(s) leading to overproduction of altered ALS enzyme at the herbicide binding site of the enzyme in lentil plant.

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