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

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Expression of an Anti-Transferrin Receptor Antibody scFV Fragment in Escherichia coli Using A L-Rhamnose-Based Tightly Regulated Promoter System Dian Andriani; Ira Handayani; Wien Kusharyoto
Annales Bogorienses Vol. 16 No. 2 (2012): Annales Bogorienses
Publisher : National Research and Innovation Agency (BRIN)

Show Abstract | Download Original | Original Source | Check in Google Scholar | DOI: 10.14203/ann.bogor.2012.v16.n2.1-6

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 heavy and light chain domains of the scFv antibody fragment was derived from the anti-TfR monoclonal antibody 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. coli rhaB promoter and corresponding 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. coli NiCo21 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 which corresponded to the predicted molecular mass based on the 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 : National Research and Innovation Agency (BRIN)

Show Abstract | Download Original | Original Source | Check in Google Scholar | DOI: 10.14203/ann.bogor.2014.v18.n2.35-44

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 tumor targeted 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 in Escherichia 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.
Generation of mCherryBody: an Anti-Transferrin Receptor Antibody Variable Fragment Linked by The Fluorescent Protein mCherry Wien Kusharyoto; Dian Andriani; Ira Handayani
Annales Bogorienses Vol. 20 No. 2 (2016): Annales Bogorienses
Publisher : National Research and Innovation Agency (BRIN)

Show Abstract | Download Original | Original Source | Check in Google Scholar | DOI: 10.14203/ann.bogor.2016.v20.n2.55-62

Abstract

A facile generation of a recombinant antibody fragment with intrinsic fluorescent properties of the monomeric mCherry fluorescent protein 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 mCherry protein. 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 mCherry fluorescent protein 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.
Co-Authors Asrul Muhamad Fuad Dian Andriani Dian Andriani Martha Sari Wien Kusharyoto