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Wien Kusharyoto

Research Center for Biotechnology, Indonesian Institute of Sciences (LIPI), Indonesia

Author-ID : 3964839

Agriculture, Biological Sciences & Forestry Biochemistry, Genetics & Molecular Biology Chemical Engineering, Chemistry & Bioengineering Chemistry Immunology & microbiology Medicine & Pharmacology

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Articles

Enhancing the Immunogenicity of Subunit Vaccines by Utilisation of Particulate Vaccine Delivery Systems Anggia Prasetyoputri; Wien Kusharyoto
ANNALES BOGORIENSES Vol 17, No 2 (2013): Annales Bogorienses
Publisher : Research Center for Biotechnology - Indonesian Institute of Sciences (LIPI)

Control and eradication of a number of infectious diseases are primarily attributed to effective vaccination programs. A concerted effort is still imperative to develop novel vaccines and improve the immunogenicity of existing ones with regards to efficacy, immunogenicity and safety. Rational design of vaccines using subunit vaccines is a potentially safer alternative to conventional vaccines, yet they are poorly immunogenic without additional adjuvant. Using antigen carriers to enhance their immunogenicity in the forms of adsorption or encapsulation with a delivery system has been widely investigated as an alternative to currently available adjuvants. This review aims to elaborate on the existing nanotechnology being used to develop more immunogenic subunit vaccines, with focus on particulate delivery systems for development of prophylactic vaccine candidates.

Expression of An Immunogenic Intimin Fragment of EHEC O157:H7 in Escherichia coli Periplasm under The Control of A Rhamnose-Based Regulated Promoter Hariyatun Hariyatun; Antonius Suwanto; Wien Kusharyoto
ANNALES BOGORIENSES Vol 18, No 1 (2014): Annales Bogorienses
Publisher : Research Center for Biotechnology - Indonesian Institute of Sciences (LIPI)

Intimin is the main adhesin of Enterohemorrhagic E. coli (EHEC) O157:H7 bacteria which are the most common leading infectious cause of bloody diarrhea and acute kidney failure in children who develop hemolytic uremic syndrome (HUS). Intimin is required for persistent bacterial colonization to eukaryotic host cell and its receptor-binding activity is localized at the C-terminus 282 amino acids (Intimin282). Thus, Intimin282 is an attractive antigen candidate that could be useful in vaccine and diagnostic systems against EHEC infections. Previous studies had reported expression of Intimin in E. coli cytoplasm using commonly used prokaryotic expression systems. However, it usually encountered several problems, i.e. low expression level, leaky expression, inclusion body formation, and truncated protein. The pRHA vector, which is tightly regulated by Lrhamnose and D-glucose, represents a viable alternative E. coli expression system to overcome such problems. Moreover, E. coli periplasm has an advantage of maintaining protein functionality by providing an oxidative environment that is more efficient than cytoplasm. However, to date there is no study about Intimin expression using pRHA expression system and/or in E. coli periplasm. Accordingly, we constructed a recombinant pRHA vector harbouring the respective gene to investigate the expression of an immunogenic Intimin fragment of EHEC O157:H7 in E. coli periplasm. The gene encoding His6-tagged Intimin282 (Int282) together with pelB signal sequence was cloned into the pRHA vector, subsequently expressed in E. coli JM109 and purified. Expression and purification of Int282 were verified by SDS-PAGE and Western blot. The result showed that Int282 was successfully expressed in E. coli periplasm with a protein size of approximately 32 kDa, which corresponded with the predicted size of the protein based on its amino acid sequence.

Preparation of An scFv-Based Immunoliposome Specific towards Transferrin Receptor Wien Kusharyoto; Ira Handayani; Martha Sari; Asrul Muhamad Fuad
ANNALES BOGORIENSES Vol 18, No 2 (2014): Annales Bogorienses
Publisher : Research Center for Biotechnology - Indonesian Institute of Sciences (LIPI)

An ideal therapeutic for cancer would be one that selectively targets to tumor cells, is nontoxic to normal cells, and that could be systemically delivered, thereby reaching metastases as well as primary tumor. Immunoliposomes directed by monoclonal antibody or its fragments are promising vehicles for tumor targeted drug delivery. Transferrin receptors (TfR) levels are elevated in various types of cancer cells and considered to correlate with the aggressive or proliferative ability of tumor cells. Therefore, TfR levels can be elaborated as a prognostic tumor marker, and TfR is a potential target for drug delivery in the therapy of malignant cells. Here, we report the preparation of an anti-TfR single-chain antibody variable (scFv) immunoliposome for tumortargeted delivery vehicle. The cDNA encoding the variable heavy and light chain domains of the anti-TfRscFv antibody fragment was derived from the murine monoclonal antibody Clone E6, which is specific towards transferrin receptor. The gene encoding the anti-TfR scFv fragment was codon optimized for expression inEscherichia coli, subsequently synthesized, and cloned into the expression vector pJexpress404. The His6- tagged anti-TfR scFv fragment was expressed in E. coli and purified by means of immobilized metal-ion affinity chromatography on TALON™ matrix. SDS-PAGE revealed that the scFv fragment had the size of approximately 27 kDa, which corresponded with the predicted size of the protein based on its amino acid sequence. Liposome containing 5% MPB-DOPE were prepared by ethanol injection method. Afterwards, the anti-TfR scFv fragments were covalently conjugated to the liposome to produce the anti-TfR scFv immunoliposome with the size of around 200 to 300 nm.

Screening for Natural Producers Capable of Producing 1,3-Propanediol from Glycerol Dian Andriani; Wien Kusharyoto; Bambang Prasetya; Thomas Wilke; Klaus Dieter Vorlop
ANNALES BOGORIENSES Vol 14, No 1 (2010): Annales Bogorienses
Publisher : Research Center for Biotechnology - Indonesian Institute of Sciences (LIPI)

Glycerol is a renewable resource found as the main by-product in the transesterification of triglycerides and fat saponification. Due to the increased production of plant oils, especially palm oil in developing countries, and their larger use by the oleochemical industry, glycerol surpluses are on the world market and this may result in a desrease in glycerol price. As a consequence, biotechnological processes have been developed to convert this substrate into value-added products, such as 1,3-propanediol (1,3-PD). The microbial production of 1,3-PD could be competitive to chemical routes assuming that it is based on cheap raw material and an optimized process. In the screening for 1,3 PD–producing bacteria, raw glycerol as by-product from rapeseed oil processing unit was used as a carbon source compared with commercial glycerol. By using increasing concentration of both glycerols from 50 to 150 g/l, two potential bacteria were obtained from soil samples.BMP 1 was obtained from an enrichment culture using 50 g/l commercial glycerol, while BMR-1 was obtained from an enrichment culture using 100 g/l raw glycerol. The highest conversion yield obtained using the isolateBMP-1 was around 0.62 g 1,3-PD formed per mol glycerol consumed, and 0.73 mol 1,3-PD formed per molgycerol using the isolate BMR-1. No bacteria were obtained from cultures using 150 g/l commercial and rawgycerol, respectively, which indicated that higher concentration of glycerol has inhibition effect. Keywords: 1,3-propanediol, enrichment culture, glycerol, palm oil, screening

Co-Authors Anggia Prasetyoputri Antonius Suwanto Asrul Muhamad Fuad Bambang Prasetya Dian Andriani Hariyatun Hariyatun Ira Handayani Klaus Dieter Vorlop Martha Sari Thomas Wilke

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