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Kusumawati, Arizah
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The Purification of Rennin-Like Protease from Lactobacillus paracasei Isolated from Ettawa Goat Milk Putranto, Wendry Setiyadi; Mustopa, Apon Zaenal; Kusumawati, Arizah; Prastyowati, Anika
Annales Bogorienses Vol. 24 No. 2 (2020): Annales Bogorienses
Publisher : BRIN

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There is a protease produced by bateria that has characteristics similar to rennin from a calf. Rennin has the ability to clot casein in milk. Rennin-like protease (RLP) is produced by bacteria extracellularly. Lactic Acid Bacteria (LAB) have the potential to be developed for RLP production because they are safe and non-pathogenic bacteria. Rennin is needed in the process of milk coagulation to subsequently obtain a curd in the process of making cheese. In this study, the LAB isolated from Ettawa goat milk (isolate 2.12) which produced RLP was 99% identical to Lactobacillus paracasei based on 16S rRNA gene sequence analysis. The purification of the RLP L. paracasei 2.12 with 60% ammonium sulfate deposition, dialysis, and filtration gel chromatography Sephadex G-50 showed a single 38 kDa protein band with SMCA/SPA was 4.48 higher than that of the calf rennet with a ratio value of 1, therefore in this study, RLP L. paracasei 2.12 was developed as an alternative to renin in cheese making.
Optimization Expression and Stability Test of Recombinant Human Interferon Alfa 2a Fusion Protein in Escherichia coli BL21 (DE3) Santoso, Adi; Kusumawati, Arizah
Annales Bogorienses Vol. 19 No. 1 (2015): Annales Bogorienses
Publisher : BRIN

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The rhIFN α2a is expressed as a fusion protein containing thioredoxine and polyhistidine sites at its N terminal. Our previous research has obtained recombinant human IFN α2a (rhIFN α2a) protein that expressed predominantly as a soluble form in E. coli BL21 (DE3). Through systematic approach of various culture conditions, the aim of current this research is to acquire the best condition and its stability of recombinant rhIFN α2a fusion protein in a culture under study. Expression optimization performed by using three parameters, i.e.: temperature, induction time and inducer concentration. Various IPTG concentrations are 0.25, 0.5, 0.75, and 1.0 mM. The incubation time of bacterial cell culture carried out in 3, 4, and 5 hours at temperature 28, 30, and 37°C. The best condition was used to analyze the stability of rhIFN α2a protein expression up to ten generation. The expressed protein was analyzed using SDS PAGE and CBB staining. The optimal culture condition was found to be 37 °C temperature with 4 hours time of induction and 1 mM IPTG concentration. Stability analysis revealed that the rhFN α2a protein expression remained stable until the tenth generation with molecular weight, approximately, 36 kDa. 
Optimizaton of Cationic Lipid Mediated Transfection of pEGFP-c1 and pJ-EPO Plasmids in Chinese Hamster Ovary (CHO) Cells Attached Culture for Transient and Stable Recombinant Human Erythropoietin (rhEPO) Expression Septisetyani, Endah Puji; Kusumawati, Arizah; Santoso, Adi
Annales Bogorienses Vol. 19 No. 1 (2015): Annales Bogorienses
Publisher : BRIN

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Cationic lipid is one of transfection agents which show high efficiency and low cytotoxicity. The transfection efficiencies can be varied depending upon the type or amount of cationic lipids, the cell line or DNA plasmid being used for transfection. The purpose of this study was to find optimal condition for transfection of CHO-K1 and CHO-S cells with pJ-EPO plasmid (containing human erythropoietin/ hEPO gene) compared with pEGFP-c1 plasmid (containing green fluorescence protein/gfp gene) by cationic lipid Lipofectamin 2000TM (lipofectamin) to generate stable transfectant expressing recombinant human erythropoietin (rhEPO). Optimization was carried out regarding the amount of lipofectamin, DNA concentration, and concentration of antibiotic Geneticin (G418) for selection of stable transfectants. By using standard amount of lipofectamin (10 μl/well) in 6-well plate, highest expression level of green fluorescent protein (GFP) was shown after transfection of CHO-K1 cells with 3 μg/well pEGFP-c1 while highest expression level of rhEPO was observed after transfection of CHO-K1 cells with 6, 8, or 10 μg/well pJ-EPO plasmid. The data also indicated that optimal transfection conditions of CHOK1 and CHO-S cells with pJ-EPO were shown with the use of 4 μg/well DNA in combination with 15 μl lipofectamin. Concentration of G418 used during cells selection also affected the expression where strongest rhEPO expression was shown at 750 ng/μl G418 concentration. Similar to GFP expression profile, rhEPO signal was detected very low during selection process sbased on Western blot data at day 9. Stronger rhEPO signal was observed after day 20 when the stable transfectants have been obtained.
In-Silico Cloning and Analysis of Divalent Subunit OMP31-SODc Proteins As A Prophylaxis Vaccine Against Brucella melitensis Infection Wijaya, Sri Kartika; Kusumawati, Arizah; Wardiana, Andri; Rubiyana, Yana; Husnaa, Ulfatul; Santoso, Adi
Annales Bogorienses Vol. 19 No. 1 (2015): Annales Bogorienses
Publisher : BRIN

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The urgency to develop a new protein subunit based vaccine candidate against Brucella was provoked by its frequent infection to human and livestock. Since Brucella melitensis is found as the most frequently isolated Brucella species from human, thus the outer membrane of B. melitensis becomes a prominent subcellular localization to search for promising antigen to be developed as vaccine candidate due to its interaction with host cell. Among outer membrane proteins suggested by Vaxign program, OMP31 was found as the most promising candidate. Moreover, analysis on other subcellular localization led our interest to SODc protein, which was expected to support OMP31 in triggering immune response. The OMP31-SODc divalent vaccine candidate was analysed in silico to predict its stable three-dimensional structure, cloning process and expectation on the ease during expression, purification and vaccine delivery to elicit the expected immune response.
Co-Authors Adi Santoso Andri Wardiana, Andri Anika Prastyowati, Anika Apon Zaenal Mustopa Endah Puji Septisetyani, Endah Puji Husnaa, Ulfatul Wendry Setiyadi Putranto Wijaya, Sri Kartika Yana Rubiyana, Yana