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Ira Handayani
Research Center 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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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 : 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: 10.14203/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 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
Co-Authors Dian Andriani Wien Kusharyoto