Preparation and identification of scFv and bsFv against transferrin receptor

Abstract

To obtain single chain variable fragment (scFv) and bivalent single chain variable fragment (bsFv) against transferrin receptor, up-stream and down-stream primers were designed according to the complementary sequences of FR1 region of variable heavy (VH) and FR4 of variable light (VL), respectively, which contained inter-linker G4S and the restriction endonuclease SfiI, AscI and NotI. Two pieces of scFv fragments were first amplified through PCR and then inserted into plasmid pAB1, which could express scFv protein once induced by IPTG in the host bacteria. To express scFv and bsFv, E. coli TG1 was cultured in LB broth and was induced by IPTG. The restriction enzyme digestion map and DNA sequencing demonstrated that scFv and bsFv genes were successfully inserted into the expression plasmid. SDS-PAGE and Western blotting revealed the protein band at 35kD and 60kD, which were consistent with the molecular weight of scFv and bsFv respectively. Flow cytometry showed that scFv and bsFv harbored the specific binding activity with TfR expressed in various tumor cells, and the avidity of bsFv was higher than that of the parent scFv.

Generation and functional characterization of the anti-transferrin receptor single-chain antibody-GAL4 (TfRscFv-GAL4) fusion protein

“…We also found that the binding rate for the TfRscFv-GAL4 fusion protein with seven different tumor cell lines varied between 8.23% and 54.11%. The differences for the binding rate may account for TfR being found expressed preferentially in the proliferation or differentiation stage of these cells rather than G0 stage, which was consistent with the results from Crepin R et al [37] and our own results previously [12]. ELISA analysis indicated that the TfRscFv-GAL4 fusion protein can bind to anti-GAL4 antibody.…”

“…A fragment for the TfRscFv cDNA was directly amplified by PCR using the amplimers 5'-CGGCCATGGCCCACGTTCAGCTGCAGCAGT-3' and 5'-GGCGGAATTCTTTGATTTCCAGCTTGGTC-3' from a pUC19 plasmid containing the TfRscFv sequence as described before [12]. The fragment for GAL4 was released from a pABgal4 plasmid (kindly provided by Dr. Bert W. O' Malley, Baylor College of Medicine, Houston, TX) by digestions of EcoRI and NotI restriction endonucleases (Thermo Scientific Fermentas).…”

“…Cell pellets were frozen at −20°C and resuspended in lysis buffer (6 M guanidine-HCl, 20 m M sodium phosphate pH 7.8, 500 m M sodium chloride), sonicated 3×, and 0.45 μm filtered to remove insoluble debris. The extract of inclusion bodies was analyzed by 12.5% polyacrylamide gel electrophoresis (SDS-PAGE) under reducing conditions described as before [2,12]. Because TfRscFv-GAL4-pET expression vectors introduce a (His) 6 tail at the C-terminus of the recombinant TfRscFv-GAL4, The solubilized fusion protein was purified by immobilized metal affinity chromatography (IMAC) through the binding of His-tag with Ni 2+ -nitrilotriacetate (Ni-NTA) agarose column (Invitrogen, USA) according to the manufacturer’s protocol as previously described [12,38] with minor modifications.…”

“…The extract of inclusion bodies was analyzed by 12.5% polyacrylamide gel electrophoresis (SDS-PAGE) under reducing conditions described as before [2,12]. Because TfRscFv-GAL4-pET expression vectors introduce a (His) 6 tail at the C-terminus of the recombinant TfRscFv-GAL4, The solubilized fusion protein was purified by immobilized metal affinity chromatography (IMAC) through the binding of His-tag with Ni 2+ -nitrilotriacetate (Ni-NTA) agarose column (Invitrogen, USA) according to the manufacturer’s protocol as previously described [12,38] with minor modifications. After the solubilized inclusion bodies were passed through the column, the column was washed with 10 column volumes of 6M guanidine, 0.1 M Tris–HCl (pH 7.0) followed by 10 bed volumes of 6M urea, 50 mM Tris–HCl, 10 mM imidazole and 20 bed volumes of 6 M urea, 50 mM Tris–HCl, 50 mM imidazole.…”

“…Indeed, many TfR-specific antibodies (mAbs) have been developed and employed to kill the malignant cells in vitro and in vivo [9-11]. Previously, we have successfully developed several antibodies against TfR including the human-mouse chimeric antibody, intracellular antibody and bivalent single chain variable fragment (bsFv) antibody [6,12-14]. All of these antibodies displayed tumor-specific biodistribution with substantial antitumor activity.…”