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The Effect Of Temperature On Recombinant Human Granulocyte Colony Stimulating Factor Production By Pichia pastoris Expression System Adiredja, Yuliawati; Fuad, Asrul Muhamad
Indonesian Journal of Pharmacy Vol 29 No 2, 2018
Publisher : Faculty of Pharmacy Universitas Gadjah Mada, Yogyakarta, Skip Utara, 55281, Indonesia

Granulocyte colony stimulating factor (G-CSF) is a haematopoetic growth factor that functions as specific stimulator of the survival, proliferation, and differentiation of precursor cells of the neutrophilic granulocyte cell lineage as well as an activator of mature neutrophil function. The main objective of this work is to compare the effect of different temperature on the production of extracellular recombinant G-CSF in Pichia pastoris. Cells were cultured for 72h in baffled shake-flasks at 20°C, 25°C, and 30°C in two different medium; buffered glycerol/methanol-complex medium (BMGY/BMMY) and buffered minimal glycerol/methanol (BMGH/BMMH) after methanol induction every 12h. Expressed recombinant hG-CSF in the methylotrophic yeast P. pastoris was analyzed with SDS-PAGE. The 23 kDa protein was secreted into the culture supernatant when induced with methanol. Production of recombinant G-CSF protein in P. pastoris at 30°C at 48h incubation after methanol induction every 12h is the highest in both complex and minimum medium.

Cloning of the GOX-Xho Gene IPBCC 08.610 into Plasmid pTA2 and Its Characterization Aryani, Hanifah; Akbar, Nadhira Fathiaz; Fuad, Asrul Muhamad; Ambarsari, Laksmi; Kurniatin, Popi Asri
Jurnal Kimia Valensi Jurnal Kimia VALENSI, Volume 10, No. 2, November 2024
Publisher : Syarif Hidayatullah State Islamic University

Show Abstract | Download Original | Original Source | Check in Google Scholar | DOI: 10.15408/jkv.v10i2.39602

Glucose oxidase (GOX) from Aspergillus niger catalyzes the oxidation of β-D-glucose to δ-gluconolactone and hydrogen peroxide, making it valuable for industrial applications. Intracellular GOX exhibits higher activity than its extracellular counterpart. This study focuses on enhancing the extracellular production of GOX through recombinant DNA technology. This study aimed to reconstruct the GOX gene by adding XhoI sites at both ends, inserting a glu-ala-glu-ala spacer at the 5' end, and introducing an XbaI site at the 3' end. These modifications facilitate the cloning of the GOX-Xho gene into the pTA2 vector and its subsequent ligation into the pPICZαB expression vector, allowing for extracellular production of GOX through fusion with the α-mating factor (α-MF) signal peptide. The GOX-Xho gene was successfully amplified, cloned, and characterized. The pTA2-GOX-Xho recombinant plasmid was verified through sequencing and restriction analysis, confirming the present and correct orientation of the 1797 bp GOX-Xho gene. However, sequencing revealed several point mutations, necessitating further computational analysis to predict their impact on the enzyme's structure and function before recombinant protein expression.

Optimization of PCR Conditions for Adding XhoI Restriction Sites to the Glucose Oxidase Gene of Aspergillus niger IPBCC 08.610 Aryani, Hanifah; Akbar, Nadhira Fathiaz; Kurniatin, Popi Asri; Fuad, Asrul Muhamad; Ambarsari, Laksmi
Current Biochemistry Vol. 11 No. 1 (2024)
Publisher : IPB University

Show Abstract | Download Original | Original Source | Check in Google Scholar | DOI: 10.29244/cb.11.1.2

Glucose oxidase (GOX) is naturally produced by fungi Aspergillus niger. The GOX enzyme catalyzes the oxidation reaction of β-D-glucose to produce δ-gluconolactone and hydrogen peroxide a (H2O2). Hydrolysis of δ-gluconolactone will produce gluconic acid. Gluconic acid and its derivatives have benefits in the pharmaceutical field as a drug for mineral deficiencies. A. niger IPBCC 08.610 is a local isolate that produce intracellular GOX with higher yield than extracellularly. GOX can be expressed extracellularly by cloning into the expression vector pPICZαB which has the signal peptide α-mating factor (α-MF). GOX gene construction needs to be done by adding some features such as XhoI restriction sites at the 5' and 3' ends, XbaI restriction site at 3’ side, and spacer peptide glu-ala-glu-ala at 5’ side. This research aims to optimize Polymerase Chain Reaction (PCR) conditions in two stages of amplification, stage I to copy the GOX gene and stage II to add those features so it is hoped that recombinant GOX can increase gluconic acid production. The results of primer concentration optimization showed that primers with a concentration of 10 µM produced clearer PCR amplicons than those with a concentration of 20 µM. The optimal temperature for amplification stage I is 58°C. The amplification stage II annealing temperature was modified with the first ten cycles based on the lowest Tm of primer value, 52°C, and the subsequent 25 cycles based on the highest Tm of primer value, 61°C.

Cloning of the GOX-Xho Gene IPBCC 08.610 into Plasmid pTA2 and Its Characterization Aryani, Hanifah; Akbar, Nadhira Fathiaz; Fuad, Asrul Muhamad; Ambarsari, Laksmi; Kurniatin, Popi Asri
Jurnal Kimia Valensi Jurnal Kimia VALENSI, Volume 10, No. 2, November 2024
Publisher : Department of Chemistry, Faculty of Science and Technology Syarif Hidayatullah Jakarta State Islamic University

Show Abstract | Download Original | Original Source | Check in Google Scholar | DOI: 10.15408/jkv.v10i2.39602

Glucose oxidase (GOX) from Aspergillus niger catalyzes the oxidation of β-D-glucose to δ-gluconolactone and hydrogen peroxide, making it valuable for industrial applications. Intracellular GOX exhibits higher activity than its extracellular counterpart. This study focuses on enhancing the extracellular production of GOX through recombinant DNA technology. This study aimed to reconstruct the GOX gene by adding XhoI sites at both ends, inserting a glu-ala-glu-ala spacer at the 5' end, and introducing an XbaI site at the 3' end. These modifications facilitate the cloning of the GOX-Xho gene into the pTA2 vector and its subsequent ligation into the pPICZαB expression vector, allowing for extracellular production of GOX through fusion with the α-mating factor (α-MF) signal peptide. The GOX-Xho gene was successfully amplified, cloned, and characterized. The pTA2-GOX-Xho recombinant plasmid was verified through sequencing and restriction analysis, confirming the present and correct orientation of the 1797 bp GOX-Xho gene. However, sequencing revealed several point mutations, necessitating further computational analysis to predict their impact on the enzyme's structure and function before recombinant protein expression.

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