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Adi Santoso
Research Center for Biotechnology, Indonesian Institute of Sciences (LIPI)
Agriculture, Biological Sciences & Forestry Biochemistry, Genetics & Molecular Biology Chemical Engineering, Chemistry & Bioengineering Chemistry Immunology & microbiology Medicine & Pharmacology

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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 Endah Puji Septisetyani; Arizah Kusumawati; Adi Santoso
ANNALES BOGORIENSES Vol 19, No 1 (2015): Annales Bogorienses
Publisher : Research Center for Biotechnology - Indonesian Institute of Sciences (LIPI)

Show Abstract | Download Original | Original Source | Check in Google Scholar | Full PDF (687.725 KB) | DOI: 10.14203/ann.bogor.2015.v19.n1.9-16

Abstract

Cationic lipid is one of transfection agents which show high efficiency and low cytotoxicity. The transfection efficiencies may depend 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 (contain human epo gene) compared with pEGFP-c1 plasmid (contain gfp gene) by cationic lipid Lipofectamin 2000TM 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. Using standard amount of lipofectamin (10µl/well) in 6-well plate, highest expression level of green fluorescent protein (GFP) was shown after transfection of CHO-K1 cells with 4µg/well pEGFP-c1 while highest expression level of rhEPO was observed after transfection of CHO-K1 cells with 6, 8, and 10µg/well pJ-EPO plasmids. The data also indicated that optimal transfection conditions of CHO-K1 and CHO-S cells with pJ-EPO were shown with the use of 4µg/well DNA and 15µl lipofectamin, respectively. Concentration of G418 affected the expression where strongest rhEPO expression was shown at 1,000 ng/µl G418 concentration. 
Optimization Expression and Stability Test of Recombinant Human Interferon Alfa 2a Fusion Protein in Escherichia coli BL21 (DE3) Adi Santoso; Arizah Kusumawati
ANNALES BOGORIENSES Vol 19, No 1 (2015): Annales Bogorienses
Publisher : Research Center for Biotechnology - Indonesian Institute of Sciences (LIPI)

Show Abstract | Download Original | Original Source | Check in Google Scholar | Full PDF (423.849 KB) | DOI: 10.14203/ann.bogor.2015.v19.n1.1-8

Abstract

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 mM, 0.5 mM, 0.75 mM 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.
Co-Authors Arizah Kusumawati Endah Puji Septisetyani