14 research outputs found
Identifikasi dan Kloning Gen CalBsyn ke dalam Plasmid pJ912-AGα-RFP di Escherichia coli
No full textSistem tampilan permukaan khamir telah menjadi teknik yang semakin
popular untuk rekayasa protein dan pembuatan pustaka protein. Protein heterolog
yang ditampilkan pada permukaan sel khamir diekspresikan dalam bentuk fusi
dengan suatu protein penahan atau protein permukaan. Protein CALB (Candida
antratica Lipase B) yang disandikan oleh gen sintetik CalBsyn dapat ditingkatkan
efektivitasnya melalui ekspresi pada permukaan sel khamir. Plasmid pJ912-AGα-
RFP mengandung gen penyandi protein permukaan, AGα (Agglutinin-α) dan gen
penyandi protein penanda ekspresi, RFP (Red fluorescent protein). Penelitian ini
bertujuan untuk memperoleh konstruksi vektor pJ912-AGα-RFP-CalBsyn untuk
ekspresi protein CALB rekombinan pada permukaan sel khamir Pichia pastoris
melalui kloning gen CalBsyn ke dalam plasmid pJ912-AGα-RFP pada Escherichia
coli. Gen CalBsyn tanpa sinyal sekresi natifnya diperoleh dari plasmid pJ912-
CALB melalui amplifikasi PCR menggunakan pasangan primer mCalB-Sal-F dan
mCalB-His-Sal-R. Gen mCalBsyn yang diperoleh berukuran panjang 1004 bp. Baik
gen mCalBsyn maupun plasmid pJ912-AGα-RFP dipotong dengan enzim restriksi
SalI, kemudian diligasi dan ditransformasi ke dalam E. coli dengan metode kejut
panas. Transformasi menghasilkan 11 koloni transforman putatif yang selanjutnya
dipurifikasi menjadi koloni-koloni tunggal. Analisis PCR dengan primer AOX1-F
dan mCalB-His-Sal-R menunjukkan adanya 34 koloni tunggal dari klon yang
dianalisis terindikasi positif membawa plasmid rekombinan pJ912-AGα-RFPCalBsyn
Bioassay of Recombinant Human Granulocyte Colony Stimulating Factor (rhG-CSF) for Neutropenia Treatment in Male Sprague Dawley Rats
No full textBackground: Recombinant human granulocyte colony stimulating factor (rhG-CSF) is a first line therapy for neutropenia. However, it is less affordable for most patients in developing and poor countries. Therefore, biosimilar products are developed to suppress the cost of treatment, namely with rhG-CSF. This study aimed to explore the establishment of an affordable rhG-CSF that has similar potential to induce neutrophils recovery as the positive control.Materials and Methods: The rhG-CSF was expressed as inclusion body in Escherichia coli NiCo21(DE3). The inclusion body was then solubilized, refolded, purified and characterized prior to be used in the bioactivity assay. Cyclophosphamide-induced male Sprague Dawley rats were used as animal model and administered with rhG-CSF. Blood sample was collected at several points of time, before and after rhG-CSF treatments. Complete blood count and peripheral blood smear were conducted to investigate the activity of the rhG-CSF on each blood cells type, particularly neutrophil.Results: Specific activity on neutrophil proliferation was shown after treatments with our rhG-CSF and positive control. Positive control dose 40 mg/kg BW was statistically similar with that of the rhG-CSF dose 80 and 120 mg/kg BW. However, in neutropenic condition, recovery of neutrophil counts could not be achieved within 4 days of treatments. Thus, a longer treatment is needed to observe the activity of the rhG-CSF as an antineutropenia agent.Conclusion: The rhG-CSF has been proven having specific activity on neutrophil proliferation. However, improvement in the rhG-CSF preparation is still needed and longer administration of the rhG-CSF has to be applied in the future study.Keywords: rhG-CSF, biosimilar, neutropenia, Sprague Dawley rat
Konstruksi Gen Glukosa Oksidase untuk Subkloning ke dalam Plasmid pPICZα dengan Penambahan Oligonukleotida Penyandi Spacer Peptida
No full textGlukosa oksidase (EC 1.1.3.4) merupakan flavoenzim yang berasal dari
Aspergillus niger. Enzim Glukosa Oksidase (GOX) intraseluler yang berasal dari
A. niger memiliki aktivitas yang lebih tinggi dibandingkan dengan aktivitas enzim
GOX ekstraselulernya. Enzim GOX intraseluler yang berasal dari A. niger memiliki
tingkat produksi yang rendah serta proses pemurnian yang cukup rumit. Penelitian
ini bertujuan untuk mendapatkan gen penyandi GOX yang siap untuk disisipkan ke
dalam plasmid pPICZα untuk kemudian diekspresikan pada Pichia pastoris secara
ekstraseluler. Perbanyakan plasmid rekombinan pGEM®-T Easy-GOX dilakukan
pada sel bakteri Escherichia coli DH5α. Konstruksi gen GOX dilakukan dengan
amplifikasi menggunakan PCR. Amplifikasi dilakukan sebanyak dua kali. Ukuran
panjang basa yang diperoleh dari hasil amplifikasi kedua sebesar 1787 bp, terdiri
atas situs restriksi XhoI dan oligonukleotida penyandi spacer peptida pada ujung 5’
sekuens gen GOX, serta situs restriksi XbaI pada ujung 3’ sekuens gen GOX
The Effect Of Temperature On Recombinant Human Granulocyte Colony Stimulating Factor Production By Pichia pastoris Expression System
No full textGranulocyte 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
Rancang Bangun vacuum Penyedot debu Sebagai fasilitas galangan Mini Jurusan teknik Perkapalan politeknik Negeri Bengkalis
No full textBengkalis State Polytechnic Mini Shipbuilding Engineering Shipyard often encounters work in the process of building fiberglass ships. Every time you carry out the manufacturing process, of course you first make a mold and when making the mold you first cut or carve the part of the plywood or board that you want to make a mold from. After carrying out the sanding or cutting process, of course the dust from the fibers is scattered on the floor. Usually this cleaning is
done using a broom because the vacuum cleaner in the yard is unstable and therefore cannot be used anymore. The dust in the workshop is dust that comes from wood fibers and fibres. In simple terms, the working principle of a vacuum cleaner is a suction and exhaust process using a number of physical principles to clean dirt effectively. Tool design is the process of creating a drawing using the AutoCAD application, where at this stage the author will design the shape of the
vacuum cleaner. As for how the vacuum cleaner works. What the author made is not much different from the vacuum cleaner sold in stores, it's just that the materials needed are different
Modification of recombinant human epidermal growth factor (rh-EGF) expression vector by site-directed mutagenesis for therapeutic protein production
No full textRecombinant human epidermal growth factor (rh-EGF) has high value in therapies for h-EGF deficiency-related diseases. The expression of the h-EGF gene was designed by using the pET21b(+) vector and Escherichia coli BL21(DE3) as the expression host. In a previous study, the sequence of a 6xHis tag without any restriction sites was fused to the h-EGF gene, yet it was not possible to obtain a purified and single rh-EGF by this approach. In this study, we modified the rh-EGF expression vector using site-directed mutagenesis (SDM) to remove the sequence of the 6xHis tag. The vector modification was carried out by inserting a stop codon and the EcoRI restriction site, along with deleting the 6xHis tag sequence. The results of PCR showed non-specific bands, while 2-step cycles PCR produced one non-specific band, and 3-step cycles PCR produced two non-specific bands. After purification of the PCR products, the SDM-recombinant plasmids treated for template plasmid-free product were transformed into E. coli DH5a. Even though the transformation efficiency was low, the planned gene mutations including the deletion of the 6xHis tag and insertion of the stop codon and EcoRI restriction site in plasmid pET21b(+) were successfully carried out. When using this modified vector in expression studies, rh-EGF of a similar size to that of the rh-EGF standard and approximately 1 kDa smaller than the rh-EGF-6xHis of the previous study was obtained
Optimization of expression JTAT protein with emphasis on transformation efficiency and IPTG concentration
No full textOptimization of expression JTAT protein with emphasis on transformation efficiency and IPTG concentration
No full textOne of small accessory genes between pol and env is tat gene encoding TAT protein. This research was aimed to optimize the expression of Jembrana TAT (JTAT) protein with preparing Escherichia coli (E. coli) in advance using adopted methods of M1 (MgCl2 + CaCl2) and M2 (CaCl2 + Glycerol). The best transformation efficiency resulting from a better transformation method was used to subsequent expression of JTAT protein. A synthetic tat gene encoding protein JTAT was previously cloned into pBT-hisC. Concentration of 200; 400; 600 µM IPTG was induced to a small volume culture (200 ml; OD600 = 4), incubated for 3 h. Pellets were harvested by centrifugation (4000 rpm; 4 °C; 15 min). Buffer B (10 mM Immidazole) was added into pellets, lysed by freeze-thaw followed by sonication. Supernatant was collected by centrifugation (10,000 rpm; 4 °C; 20 min) and purified using Ni-NTA Agarose resin, released by elution buffer (E) containing 400 mM Immidazole to collect purified protein twice (E1, E2). The protein was characterized by SDS-PAGE and Western Blot (WB), quantified (at λ595 nm) with BSA standard method in prior. The result showed that transformation efficiency was better in M2 (2.53 × 106) than M1 (3.10 × 105). The JTAT protein was expressed at a right size of 11.8 kDa. Concentration of 200 µM IPTG produced a significantly better protein yield (1.500 ± 0.089 mg/ml; P < 0.05) than 600 µM IPTG (0.896 ± 0.052 mg/ml) and not different to 400 µM IPTG (1.298 ± 0.080 mg/ml). This research indicated that transformation efficiency needs to be taken account in prior of optimization of the protein expression
Expression and characterization of Trichoderma reesei endoglucanase II in Pichia pastoris under the regulation of the GAP promoter
No full textTrichoderma reesei is known to be one of the organisms capable for producing various types of cellulase in high concentrations. Among these cellulases, the highest catalytic efficiency of endoglucanases II (EGII, EC 3.2.1.4) are considered important for industrial application. The characterization of the EGII is necessary since it is widely used in high-temperature reactions in the industries. In this study, the recombinant EGII protein was expressed in Pichia pastoris and it has a molecular mass of approximately 52 kDa. Recombinant EGII was purified using Ni-NTA affinity chromatography and characterized by SDS-PAGE and western blot analyses. The enzyme activity of recombinant EGII was measured using the Nelson Somogyi method to determine its optimum pH and temperature. The result showed that the maximum EGII expression was achieved after 72 h of culture incubation. The crude enzyme has optimum activity at pH 5.0, resulting in 16.3 U/mL and 14.6 U/mL activity at 40 °C and 50 °C, respectively. While the purified enzyme gave the specific activity of 115.7 U/mg under the optimum condition. Finally, our study demonstrated that recombinant EGII could retain the endoglucanase activity for 89% and 80% at 40 °C and 50 °C, respectively
Optimization of PCR Conditions for Adding XhoI Restriction Sites to the Glucose Oxidase Gene of Aspergillus niger IPBCC 08.610
No full textGlucose 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\u27 and 3\u27 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
