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Kusharyoto, Wien
Research Center for Biotechnology - Indonesian Institute of Sciences (LIPI)
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Generation of mCherryBody: an Anti-Transferrin Receptor Antibody Variable Fragment Linked by The Fluorescent Protein mCherry Kusharyoto, Wien; Andriani, Dian; Handayani, Ira
ANNALES BOGORIENSES Vol 20, No 2 (2016): Annales Bogorienses
Publisher : Research Center for Biotechnology - Indonesian Institute of Sciences (LIPI)

Show Abstract | Download Original | Original Source | Check in Google Scholar | DOI: 10.14203/ab.v20i2.233

Abstract

A facile generation of a recombinant antibody fragment with intrinsic fluorescent properties of the monomeric fluorescent protein mCherry is described. The so-called mCherryBody was designed based on the structure model of the variable fragment of anti-transferrin receptor antibody LUCA31 and the X-ray crystallographic structure of the protein mCherry. mCherryBody was constructed to retain optimal spatial geometry between the C- and N-termini of the antibody light-chain (VL) and heavy-chain (VH) by mimicking the domains interface pairing in antibody Fab fragments and incorporation of the fluorescent protein mCherry as a bridging scaffold. The gene encoding the chimeric protein was cloned into the pJExpress414 expression vector, expressed and secreted into the periplasm of Escherichia coli NiCo21(DE3) for assembly and disulphide bond formation. Based on its amino acid sequence, mCherryBody was predicted to have a molecular weight of 51.46 kDa. The modular assembly used in the generation of mCherryBody may permit the interchange of binding sites and of fluorescent proteins to create robust panels of coloured antibody fragments. Thus, the mCherryBody platform facilitates rapid generation of colored single-chain variable fragment (scFv) chimeras that could be used for screening of antibodies against cell surface markers or receptors.
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; Suwanto, Antonius; Kusharyoto, Wien
ANNALES BOGORIENSES Vol 18, No 1 (2014): Annales Bogorienses
Publisher : Research Center for Biotechnology - Indonesian Institute of Sciences (LIPI)

Show Abstract | Download Original | Original Source | Check in Google Scholar | DOI: 10.1234/90

Abstract

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 Kusharyoto, Wien; Handayani, Ira; Sari, Martha; Fuad, Asrul Muhamad
ANNALES BOGORIENSES Vol 18, No 2 (2014): Annales Bogorienses
Publisher : Research Center for Biotechnology - Indonesian Institute of Sciences (LIPI)

Show Abstract | Download Original | Original Source | Check in Google Scholar | Full PDF (664.3 KB) | DOI: 10.1234/99

Abstract

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

Show Abstract | Download Original | Original Source | Check in Google Scholar | DOI: 10.1234/43

Abstract

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
Effect of culture conditions on phytase production by Aspergillus ficuum in solid-state fermention using rice bran as substrate Kusharyoto, Wien; Sari, Martha; Ridwanuloh, Asep Muhammad
ANNALES BOGORIENSES Vol 13, No 1 (2009): Annales Bogorienses
Publisher : Research Center for Biotechnology - Indonesian Institute of Sciences (LIPI)

Show Abstract | Download Original | Original Source | Check in Google Scholar | Full PDF (259.142 KB) | DOI: 10.1234/23

Abstract

Phytic acid is an antinutritional factor that forms 1–2% of most of the seeds and their co-products representing more than 60%  of their total phosphorus. Monogastric and agastric animals are unable to utilize phytate phosphorus either due to lack of or insufficient amount of phytate degrading enzymes. Phytases (myo-inositol hexakisphosphate-phosphohydrolase) are a special class of phosphatases that catalyze the hydrolysis of phytic acid in a stepwise manner to lower inositol phosphates, myo-inositol and inorganic phosphate. Phytases are found naturally in plants and microorganisms and a sizeable number of phytases have been purified and characterized from various fungi, yeasts and bacteria. The present investigation involves studies on the effect of moisture content, pH value and different media ingredients such as carbon, nitrogen, and surfactants on the production of phytase by the fungus  Aspergillus ficuum DSM 932 in solid-state fermentation (SSF) using rice bran as substrate. The production of phytase by SSF was favored, when the fungus was grown at a moisture content of 60% and pH 7.0, resulted in a phytase activity of 5.2 units/g dry substrate. There was a 20% increase in phytase yield in the presence of sucrose in SSF medium, while glucose and fructose were not effective in enhancing the phytase activity when used individually. Yeast extract was found to be a favorable nitrogen source for phytase production by SSF, which resulted in a 20% increase in phytase activity. There was no significant effect in increasing phytase production with the use of either soy peptone or tryptic  soy as nitrogen source. Approximately 30% inhibition in phytase activity was shown in the presence of the surfactant Tween-80 or Triton X-100 in the SSF. By supplementing rice bran with sucrose and yeast extract, and performing the SSF in tray bioreactors, a phytase activity of 6.76 units/g dry substrate could be obtained.Keywords:  phytase, solid-state fermentation, Aspergillus ficuum, nutritional factors, rice bran
Enhancing the Immunogenicity of Subunit Vaccines by Utilisation of Particulate Vaccine Delivery Systems Prasetyoputri, Anggia; Kusharyoto, Wien
ANNALES BOGORIENSES Vol 17, No 2 (2013): Annales Bogorienses
Publisher : Research Center for Biotechnology - Indonesian Institute of Sciences (LIPI)

Show Abstract | Download Original | Original Source | Check in Google Scholar | Full PDF (543.649 KB) | DOI: 10.1234/76

Abstract

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.
Sensitivity Improvement of A Direct Competitive Elisa for Atrazine by Exploiting Low Cross-Reactivity of An Atrazine-Specific Recombinant Antibody Fab-Fragment Kusharyoto, Wien; Schmid, Rolf D.
ANNALES BOGORIENSES Vol 9, No 1 (2004): Annales Bogorienses
Publisher : Research Center for Biotechnology - Indonesian Institute of Sciences (LIPI)

Show Abstract | Download Original | Original Source | Check in Google Scholar | Full PDF (3557.578 KB) | DOI: 10.1234/2

Abstract

The hapten-binding site of the antibody Fab-fragment K411 B specific towards the herbicide atrazine (2-cllloro-4-(ethylamino)-6-(isopropyJamjno)- 1,3,5-triazine) was modified by means of structural modeling and site-directed mutagenesis. A triple mutant (GlnL89GlulValH3711e/GluL3Val) of the Fab-fragment showed an increased affinity towards the hapten HlCVC6 (4-arnino-6-chloro-l ,3,5-triazine-2-(6-aminohexanecarboxylic acid» compared to the affinity ofthe wild-type Fab-fragment lowards the same hapten. However, the mutant exhibited substantially lower affinity towards the hapten H/CVC6 Ihan towards atrazine and the hapten iPr/ CVC6 (4-chloro-6-(isopropylamino)-1,3,5-triazioe-2-(6-aminohexanecarboxylic acid», which is usual ly used in the synthesis of enzyme tracers in ELISA for atrazine. Advantage was taken of the low cross-reactivity and increased affinity of the mutant Fab fragment towards H/CVC6 to improve the sensitivity of a direct-competitive ELISA tor atrazine. HlCIlC6 was covalently conjugated with horseradish peroxidase (HRP), and the conjugate H/CI/C6-HRPwas used as enzyme tracer in the ELISA for atrazine. An eight-fold improvement in sensitivity ofa direct-competitive ELISA for atrazine could be achieved using the tracer H/CVC6-HRP compared to the sensitivity of ELISA using the tracer iPr/CVC6-HRP. The detection limit for atrazine was as low as 0.01 i g/l. 
Expression of an Anti-Transferrin Receptor Antibody SCFV Fragment in Escherichia coli Using A L-Rhamnose-Based Tightly Regulated Promoter System Andriani, Dian; Handayani, Ira; Kusharyoto, Wien
ANNALES BOGORIENSES Vol 16, No 2 (2012): Annales Bogorienses
Publisher : Research Center for Biotechnology - Indonesian Institute of Sciences (LIPI)

Show Abstract | Download Original | Original Source | Check in Google Scholar | Full PDF (3611.759 KB) | DOI: 10.1234/63

Abstract

Transferrin receptor (TfR) levels are elevated in various types of cancer cells and correlate with the aggressive or proliferative ability of tumor cells. Therefore, TfR levels are considered useful as a prognostic tumor marker, and TfR is a potential target for drug delivery in therapy of malignant cells. In such kind of targeted delivery system, antibody fragments are frequently used as targeting moiety. Here, we report the generation of an anti-TfR single-chain antibody variable (scFv). The cDNA encoding the variable of heavy and light chain domains of the scFv antibody fragment was derived from the anti-TfR monoclonal antibody of LUCA31. The gene encoding the anti-TfR scFv fragment was codon, optimized for expression in Escherichia coli, subsequently synthesized, and cloned into the pJExpress-804 Rhamex vector. The expression vector utilizes the E. colirhaB promoter and corresponds to the regulatory genes, and is tightly regulated by the presence of L-L-rhamnose. It is also tightly regulated in the absence of L-L-rhamnose by the addition of D-glucose The His6-tagged anti-TfR scFv fragment was expressed in E. coliNiCo21 and purified by means of immobilized metal chelate affinity chromatography on TALON™ matrix. In SDS-PAGE, a single band corresponding to a molecular mass of approximately 30 kDa was observed whether it corresponded to the predicted molecular mass based on the amino acid sequence.Keywords: antibody fragment, L-L-rhamnose, Rhamex vector, scFv, transferrin receptor
Subcloning, Expression and Characterisation of A Recombinant Antibody Fab-Fragment Specific Towards 2,4-D Kusharyoto, Wien
ANNALES BOGORIENSES Vol 10, No 1 (2005): Annales Bogorienses
Publisher : Research Center for Biotechnology - Indonesian Institute of Sciences (LIPI)

Show Abstract | Download Original | Original Source | Check in Google Scholar | Full PDF (3633.744 KB) | DOI: 10.1234/15

Abstract

A generic strategy was established for subcloning the VH and VL gene of antibody variable domains into the plasmid pASK85 for the expression of Fab antibody fragments. pASK85 bears coding sequences for murine constant domains including a His6-tag at the carboxy-terminal end of the constant heavy-chain domain. The VH and YL gene derived from the monoclonal antibody E2/B5 specific towards 2,4-dichloropbcnoxyacetic acid (2,4- D) were used in this study. Eschericia coli was used as host cells for the biosynthesis of the Fab-fragment. The Fab­ fragment wa subsequently purified from the periplasmic extract in a single step by immobilised metal-ion affinity chromatography (IMAC). The production level obtained were 0.5-0.8 mg purified Fab-fragments per liter E. coli culture. The sensitivity and cross-reactivity of the Fab-fragment determined by direct competitive ELISA were similar to those of the parental monoclonal antibody E2/B5 .  
Molecular Docking and Molecular Dynamics Study of Alcoholic Compounds as Mycobactericidal Agents Using InhA, MabA and PanK as Receptors Syahputra, Gita; Arwansyah, Arwansyah; Kusharyoto, Wien
ANNALES BOGORIENSES Vol 22, No 2 (2018): Annales Bogorienses
Publisher : Research Center for Biotechnology - Indonesian Institute of Sciences (LIPI)

Show Abstract | Download Original | Original Source | Check in Google Scholar | DOI: 10.14203/ann.bogor.2018.v22.n2.101-115

Abstract

     Tuberculosis (TB) infection is one of the primaryinfectious diseases in many developing countries; even there are minor cases in some developed countries. TB infection spread through the air and ismore probable when using improper disinfectant on medical and laboratory equipment which related to TB research. The appropriate disinfectants which are commonly usedin laboratory equipment can reduce the risk of transmission of TB disease. Alcoholic compoundsare one of the common disinfectants with a broad spectrum activity towardsmicrobes,viruses, and fungus. We employed molecular docking and molecular dynamics simulation to support virtual screening and ligand-receptor complex binding observation in searching for an appropriate mycobactericidal agent.Based on the analysis of molecular docking and molecular dynamics, pentadecanol has potency as a mycobactericidal agent with PanK as itsspecific receptor. The Gibbs free energy (∆G) for the interaction of pentadecanol with PanKhas been found to be -5.5 kcal/mol. Molecular dynamics analysis at 300K and 1 atm for 5 ns showed a little change in the confirmation of the binding site, whilepentadecanolwas still being bound by its binding siteon PanK.
Co-Authors Antonius Suwanto dan Meity S. Sinaga . Budi Tjahjono Andi Khaeruni R Arwansyah Arwansyah, Arwansyah Asrul Muhamad Fuad, Asrul Muhamad Bambang Prasetya Dian Andriani, Dian Gita Syahputra Handayani, Ira Hariyatun, Hariyatun Martha Sari Prasetyoputri, Anggia Ridwanuloh, Asep Muhammad Schmid, Rolf D. Vorlop, Klaus Dieter Wilke, Thomas