scholarly journals A Host-Vector System for the Expression of a Thermostable Bacterial Lipase in a Locally Isolated Meyerozyma guilliermondii SMB

2020 ◽  
Vol 8 (11) ◽  
pp. 1738
Author(s):  
Abu Bakar Salleh ◽  
Siti Marha Baharuddin ◽  
Raja Noor Zaliha Raja Abd Rahman ◽  
Thean Chor Leow ◽  
Mahiran Basri ◽  
...  
Keyword(s):  
Expression System ◽  
Vector System ◽  
Selection Marker ◽  
Hygromycin B ◽  
Gene Encoding ◽  
Methanol Induction ◽  
New Host ◽  
Yeast Expression System ◽  
Meyerozyma Guilliermondii ◽  
New Yeast

Screening for a new yeast as an alternative host is expected to solve the limitations in the present yeast expression system. A yeast sample which was isolated from the traditional food starter ‘ragi’ from Malaysia was identified to contain Meyerozyma guilliermondii strain SMB. This yeast-like fungus strain SMB was characterized to assess its suitability as an expression host. Lipase activity was absent in this host (when assayed at 30 °C and 70 °C) and Hygromycin B (50 μg/mL) was found to be its best selection marker. Then, the hyg gene (Hygromycin B) was used to replace the sh ble gene (Zeocin) expression cassette in a Komagataella phaffii expression vector (designated as pFLDhα). A gene encoding the mature thermostable lipase from Bacillus sp. L2 was cloned into pFLDhα, followed by transformation into strain SMB. The optimal expression of L2 lipase was achieved using YPTM (Yeast Extract-Peptone-Tryptic-Methanol) medium after 48 h with 0.5% (v/v) methanol induction, which was 3 times faster than another K. phaffii expression system. In conclusion, a new host-vector system was established as a platform to express L2 lipase under the regulation of PFLD1. It could also be promising to express other recombinant proteins without inducers.

scholarly journals Determination of Putative Vacuolar Proteases, PEP4 and PRB1 in a Novel Yeast Expression Host Meyerozyma guilliermondii Strain SO Using Bioinformatics Tools

2022 ◽  
Vol 30 (1) ◽  
pp. 777-797
Author(s):  
Okojie Eseoghene Lorrine ◽  
Raja Noor Zaliha Raja Abd. Rahman ◽  
Joo Shun Tan ◽  
Raja Farhana Raja Khairuddin ◽  
Abu Bakar Salleh ◽  
...  
Keyword(s):  
Proteolytic Activity ◽  
Heterologous Protein ◽  
Expression System ◽  
Methylotrophic Yeast ◽  
Yeast Expression ◽  
Heterologous Protein Expression ◽  
Evolutionary Analysis ◽  
Heterologous Proteins ◽  
Yeast Expression System ◽  
Meyerozyma Guilliermondii

Meyerozyma guilliermondii strain SO, a newly isolated yeast species from spoilt orange, has been used as a host to express the recombinant proteins using methylotrophic yeast promoters. However, as a novel yeast expression system, the vacuolar proteases of this yeast have not been determined, which may have contributed to the low level of heterologous protein secretions. Thus, this study aimed to determine intra- and extracellular proteolytic activity and identify the putative vacuolar proteases using bioinformatics techniques. A clear zone was observed from the nutrient agar skimmed milk screening plate. Proteolytic activity of 117.30 U/ml and 75 U/ml were obtained after 72 h of cultivation for both extracellular and intracellular proteins, respectively. Next, the Hidden Markov model (HMM) was used to detect the presence of the vacuolar proteases (PEP4 and PRB1) from the strain SO proteome. Aspartyl protease (PEP4) with 97.55% identity to Meyerozyma sp. JA9 and a serine protease (PRB1) with 70.91% identity to Candida albicans were revealed. The homology with other yeast vacuolar proteases was confirmed via evolutionary analysis. PROSPER tool prediction of cleavage sites postulated that PEP4 and PRB1 might have caused proteolysis of heterologous proteins in strain SO. In conclusion, two putative vacuolar proteases (PEP4 and PRB1) were successfully identified in strain SO. Further characterization can be done to understand their specific properties, and their effects on heterologous protein expression can be conducted via genome editing.

scholarly journals An Expression System for the E. coli Fermentation of Recombinant Antibody Fab Fragments from Mice and Rabbits

2010 ◽  
Vol 93 (1) ◽  
pp. 80-88 ◽  
Author(s):  
Julia C Wiebe ◽  
Carolin Schüller ◽  
Jana A Reiche ◽  
Karl Kramer ◽  
Arne Skerra ◽  
...  
Keyword(s):  
Recombinant Antibody ◽  
Expression System ◽  
Coli Strain ◽  
Vector System ◽  
Light Chains ◽  
Selection Marker ◽  
Specific Class ◽  
Fab Fragment ◽  
E Coli ◽  
Fab Fragments

Abstract A mutually compatible vector system was developed for the bench-top fermenter production of mouse and rabbit Fab fragments comprising pASK85-pro-K411B and pASK85Rab-pro-WR13. These vectors provide a mouse- and rabbit-specific cloning site, respectively, the tetA promoter/operator and the proBA operon that complements the Pro biosynthetic deficiency of the Escherichia coli strain JM83 and can serve as an additional selection marker. Fermentation at elevated cell density (OD600 = 2040) of the atrazine-specific mouse Fab fragment K411B using JM83 harboring pASK85-pro-K411B in a 2 L bench-top vessel resulted in a yield of 240 g/L OD600 affinity-purified protein (13.8 mg). In contrast, expression of leporid Fab fragments using pASK85Rab-pro-WR13 was unsuccessful due to the aggregation of rabbit light chains, which probably relates to a general problem of this specific class of immunoglobulins with their additional Cys residues. Coexpression of rabbit Fab fragments together with four periplasmic folding-helper proteins encoded on pTUM4 led to a significantly improved folding efficiency, resulting in a yield of 50 µg/L OD600 affinity-purified rabbit Fab fragment (3.3 mg) from the 2 L bench-top fermenter.

Hansenula polymorpha as a novel yeast system for the expression of heterologous genes

1988 ◽  
Vol 16 (6) ◽  
pp. 1081-1083 ◽  
Author(s):  
P. E. SUDBERY ◽  
M. A. GLEESON ◽  
R. A. VEALE ◽  
A. M. LEDEBOER ◽  
M. C. M. ZOETMULDER
Keyword(s):  
Linkage Group ◽  
Genetic Analysis ◽  
Hansenula Polymorpha ◽  
Expression System ◽  
Carbon Sources ◽  
Neomycin Phosphotransferase ◽  
Yeast Expression System ◽  
Methanol Oxidase ◽  
Complementation Groups ◽  
New Yeast

Summary The advantages of Hansenula polymorpha as a new yeast expression system are discussed in terms of the powerful and regulatable methanol oxidase promoter and the organism's ability to grow on cheap carbon sources. The development of techniques for conventional genetic analysis is described. A total of 218 mutants have been assigned to 62 complementation groups, three genes have been found to be linked forming the first linkage group in this organism. Methods for molecular transformation have been developed allowing the expression of heterologous genes. The disruptive integration and expression of the neomycin phosphotransferase is described.

scholarly journals Locally isolated yeasts from Malaysia: identification, phylogenetic study and characterization.

2012 ◽  
Vol 59 (2) ◽  
Author(s):  
Siti Nurbaya Oslan ◽  
Abu Bakar Salleh ◽  
Raja Noor Zaliha Raja Abd Rahman ◽  
Mahiran Basri ◽  
Adam Leow Thean Chor
Keyword(s):  
Lipolytic Activity ◽  
Expression System ◽  
Its Region ◽  
Extracellular Proteins ◽  
Vector System ◽  
Phylogenetic Study ◽  
Thermostable Lipase ◽  
Rdna Its ◽  
Rdna Its Region ◽  
New Yeast

Yeasts are a convenient platform for many applications. They have been widely used as the expression hosts. There is a need to have a new yeast expression system to contribute the molecular cloning demands. Eight yeast isolates were screened from various environment sources and identified through ribosomal DNA (rDNA) Internal Transcribed Spacer (ITS). Full sequence of the rDNA ITS region for each isolate was BLASTed and phylogenetic study was constructed by using MEGA4. Among the isolates, isolate WB from 'ragi' (used to ferment carbohydrates) could be identified as a new species in order Saccharomycetales according to rDNA ITS region, morphology and biochemical tests. Isolate SO (from spoiled orange), RT (rotten tomato) and RG (different type of 'ragi') were identified as Pichia sp. Isolates R1 and R2, S4 and S5 (from the surrounding of a guava tree) were identified as Issatchenkia sp. and Hanseniaspora sp., respectively. Geneticin, 50 µg/mL, was determined to be the antibiotic marker for all isolates excepted for isolates RT and SO which used 500 µg/mL and 100 µg/mL Zeocin, respectively. Intra-extracellular proteins were screened for lipolytic activity at 30°C and 70°C. Thermostable lipase activity was detected in isolates RT and R1 with 0.6 U/mg and 0.1 U/mg, respectively. In conclusion, a new yeast-vector system for isolate WB can be developed by using phleomycin or geneticin as the drugs resistance marker. Moreover, strains RT and R1 can be investigated as a novel source of a thermostable lipase.

scholarly journals Construction of a Vitreoscilla Hemoglobin Promoter-Based Tunable Expression System for Corynebacterium glutamicum

Catalysts ◽  
2018 ◽  
Vol 8 (11) ◽  
pp. 561 ◽  
Author(s):  
Kei-Anne Baritugo ◽  
Hee Taek Kim ◽  
Mi Na Rhie ◽  
Seo Young Jo ◽  
Tae Uk Khang ◽  
...  
Keyword(s):  
Corynebacterium Glutamicum ◽  
Fluorescent Protein ◽  
Expression System ◽  
Vitreoscilla Hemoglobin ◽  
Aminobutyric Acid ◽  
Vector System ◽  
Efficient Production ◽  
Gamma Aminobutyric Acid ◽  
Recombinant Strains ◽  
Gaba Production

Corynebacterium glutamicum is an industrial strain used for the production of valuable chemicals such as L-lysine and L-glutamate. Although C. glutamicum has various industrial applications, a limited number of tunable systems are available to engineer it for efficient production of platform chemicals. Therefore, in this study, we developed a novel tunable promoter system based on repeats of the Vitreoscilla hemoglobin promoter (Pvgb). Tunable expression of green fluorescent protein (GFP) was investigated under one, four, and eight repeats of Pvgb (Pvgb, Pvgb4, and Pvgb8). The intensity of fluorescence in recombinant C. glutamicum strains increased as the number of Pvgb increased from single to eight (Pvgb8) repeats. Furthermore, we demonstrated the application of the new Pvgb promoter-based vector system as a platform for metabolic engineering of C. glutamicum by investigating 5-aminovaleric acid (5-AVA) and gamma-aminobutyric acid (GABA) production in several C. glutamicum strains. The profile of 5-AVA and GABA production by the recombinant strains were evaluated to investigate the tunable expression of key enzymes such as DavBA and GadBmut. We observed that 5-AVA and GABA production by the recombinant strains increased as the number of Pvgb used for the expression of key proteins increased. The recombinant C. glutamicum strain expressing DavBA could produce higher amounts of 5-AVA under the control of Pvgb8 (3.69 ± 0.07 g/L) than the one under the control of Pvgb (3.43 ± 0.10 g/L). The average gamma-aminobutyric acid production also increased in all the tested strains as the number of Pvgb used for GadBmut expression increased from single (4.81–5.31 g/L) to eight repeats (4.94–5.58 g/L).

scholarly journals Discovery and evaluation of a novel step in the flavonoid biosynthesis pathway regulated by F3H gene using a yeast expression system

2019 ◽  
Author(s):  
Rahmatullah Jan ◽  
Sajjad Asaf ◽  
Sanjita Paudel ◽  
Sangkyu Lee ◽  
Kyung-Min Kim
Keyword(s):  
Western Blotting ◽  
Rice Plant ◽  
Expression System ◽  
Plant Secondary Metabolites ◽  
Flavonoid Biosynthesis ◽  
Yeast Expression ◽  
List Type ◽  
Biosynthesis Pathway ◽  
Yeast Expression System ◽  
Flavonoid Biosynthesis Pathway

AbstractKaempferol and quercetin are the essential plant secondary metabolites that confer huge biological functions in the plant defense system. These metabolites are produced in low quantities in plants, therefore engineering microbial factory is a favorable strategy for the production of these metabolites. In this study, biosynthetic pathways for kaempferol and quercetin were constructed in Saccharomyces cerevisiae using naringenin as a substrate. The results elucidated a novel step for the first time in kaempferol and quercetin biosynthesis directly from naringenin catalyzed by flavonol 3-hydroxylase (F3H). F3H gene from rice was cloned into pRS42K yeast episomal plasmid (YEP) vector using BamH1 and Xho1 restriction enzymes. We analyzed our target gene activity in engineered and in empty strains. The results were confirmed through TLC followed by Western blotting, nuclear magnetic resonance (NMR), and LC-MS. TLC showed positive results on comparing both compounds extracted from the engineered strain with the standard reference. Western blotting confirmed lack of Oryza sativa flavonol 3-hydroxylase (OsF3H) activity in empty strains while high OsF3H expression in engineered strains. NMR spectroscopy confirmed only quercetin, while LCMS-MS results revealed that F3H is responsible for naringenin conversion to both kaempferol and quercetin. These results concluded that rice F3H catalyzes naringenin metabolism via hydroxylation and synthesizes kaempferol and quercetin.HighlightsCurrent study is a discovery of a novel step in flavonoid biosynthesis pathway of rice plant.In this study F3H gene from rice plant was functionally expressed in yeast expression system.Results confirmed that, F3H gene is responsible for the canalization of naringenin and converted into kaempferol and quercetin.The results were confirmed through, western blotting, TLC, HPLC and NMR analysis.

Generation of an agonistic binding site for blockers of the M3 muscarinic acetylcholine receptor

2008 ◽  
Vol 412 (1) ◽  
pp. 103-112 ◽  
Author(s):  
Doreen Thor ◽  
Angela Schulz ◽  
Thomas Hermsdorf ◽  
Torsten Schöneberg
Keyword(s):  
Ligand Binding ◽  
Binding Site ◽  
G Protein ◽  
Binding Sites ◽  
Expression System ◽  
Muscarinic Acetylcholine Receptor ◽  
Intracellular Loop ◽  
Inverse Agonists ◽  
Yeast Expression System ◽  
High Affinity

GPCRs (G-protein-coupled receptors) exist in a spontaneous equilibrium between active and inactive conformations that are stabilized by agonists and inverse agonists respectively. Because ligand binding of agonists and inverse agonists often occurs in a competitive manner, one can assume an overlap between both binding sites. Only a few studies report mutations in GPCRs that convert receptor blockers into agonists by unknown mechanisms. Taking advantage of a genetically modified yeast strain, we screened libraries of mutant M3Rs {M3 mAChRs [muscarinic ACh (acetylcholine) receptors)]} and identified 13 mutants which could be activated by atropine (EC50 0.3–10 μM), an inverse agonist on wild-type M3R. Many of the mutations sensitizing M3R to atropine activation were located at the junction of intracellular loop 3 and helix 6, a region known to be involved in G-protein coupling. In addition to atropine, the pharmacological switch was found for other M3R blockers such as scopolamine, pirenzepine and oxybutynine. However, atropine functions as an agonist on the mutant M3R only when expressed in yeast, but not in mammalian COS-7 cells, although high-affinity ligand binding was comparable in both expression systems. Interestingly, we found that atropine still blocks carbachol-induced activation of the M3R mutants in the yeast expression system by binding at the high-affinity-binding site (Ki ∼10 nM). Our results indicate that blocker-to-agonist converting mutations enable atropine to function as both agonist and antagonist by interaction with two functionally distinct binding sites.

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.

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