Dataset Information

Construction of non-covalent single-chain Fv dimers for hepatocellular carcinoma and their biological functions.

ABSTRACT: AIM:To create new diabodies with improved binding activity to antigen of the variable light - variable heavy (VH-VL) oriented single-chain Fv dimers genes (scFv). METHODS:The linker between VH and VL genes was shortened to 3-5 amino acid residues and cloned into the vector pCANTAB5E. The recombinant plasmids were transformed into TG1 cells and sequenced. The positive transformed cells were infected by M13K07 helper phage to form human recombinant phage antibodies. Expressed products were identified by SDS-PAGE, Western blotting, size exclusion gel chromatography (SEC), ELISA and immunohistochemistry. RESULTS:Three scFv (scFv-3, scFv-4, scFv-5) were constructed successfully with binding ability to hepatocellular carcinoma 3.5-6 fold greater than their parental scFv. The single-chain Fv dimer (scFv-5, termed BDM3) with the best binding ability was successfully expressed in Yeast pichlia, as shown by. SDS-PAGE and Western blotting. SEC results suggested the molecular weight of the expressed products was about 61 kDa. Expressed products showed significantly stronger binding to hepatocellular carcinoma cells than scFv, still having 50% binding activity even after 16 h incubation as 37°C. The purified dimers were bound specifically to the tumor antigen of HCC. CONCLUSION:we have generated scFv dimers by shortening a series of linkers to 3-5 amino acid residues in VH-linker-VL orientation, resulting in highly stable and affinity-improved dimeric molecules. These will become an attractive targeting moiety in immunotherapeutic and diagnostic applications for HCC.

SUBMITTER: Bie CQ

PROVIDER: S-EPMC2998965 | biostudies-literature | 2010 May

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Construction of non-covalent single-chain Fv dimers for hepatocellular carcinoma and their biological functions.

Bie Cai-Qun CQ Yang Dong-Hua DH Liang Xu-Jing XJ Tang Shao-Hui SH

World journal of hepatology 20100501 5

<h4>Aim</h4>To create new diabodies with improved binding activity to antigen of the variable light - variable heavy (VH-VL) oriented single-chain Fv dimers genes (scFv).<h4>Methods</h4>The linker between VH and VL genes was shortened to 3-5 amino acid residues and cloned into the vector pCANTAB5E. The recombinant plasmids were transformed into TG1 cells and sequenced. The positive transformed cells were infected by M13K07 helper phage to form human recombinant phage antibodies. Expressed produc ...[more]

PMID: 21160994

Similar Datasets

Project description:Single chain Fvs (scFvs) are widely applied in research, diagnostics and therapeutic settings. Display and selection from combinatorial libraries is the main route to their discovery and many factors influence the success of this process. They exhibit low thermodynamic stability, resulting in low levels of premature cytosolic folding or aggregation which facilitates sec YEG-mediated translocation and phage in E. coli. However, there is little data analysing how this is related to and influenced by scFv protein expression.We characterised the relationship between overall scFv expression and display propensity for a panel of 15 anti-tetanus toxin scFvs and found a strong positive correlation (Rho = 0.88, p < 0.005) between the two parameters. Display propensity, overall expression and soluble localisation to the periplasm and extracellular fractions were clone specific characteristics which varied despite high levels of sequence homology. There was no correlation between display of scFv or its expression in non-fused (free) form with soluble scFv localisation to the periplasm or culture supernatant. This suggests that divergence in the fate of scFv-pIII and non-fused scFv after translocation to the periplasm accounts for the observed disparity. Differential degrees of periplasmic aggregation of non-fused scFv between clones may affect the partitioning of scFv in the periplasm and culture supernatant abrogating any correlation. We suggest that these factors do not apply to the scFv-pIII fusion since it remains anchored to the bacterial inner membrane as part of the innate phage packaging and budding process.We conclude that in the absence of premature cytosolic aggregation or folding, the propensity of a scFv to be displayed on phage is directly related to its overall expression level and is thus indirectly influenced by factors such as codon bias, mRNA abundance or putative DNA motifs affecting expression. This suggests that scFvs capable of high overall expression and display levels may not produce high yields of non phage-fused soluble protein in either the periplasmic or extracellular fractions of E. coli. This should be considered when screening clones selected from combinatorial libraries for further study.

Project description:BackgroundMost Yersinia pestis strains are known to express a capsule-like antigen, fraction 1 (F1)(.) F1 is encoded by the caf1 gene located on the large 100-kb pFra plasmid, which is found in Y. pestis but not in closely related species such as Yersinia enterocolytica and Yersinia pseudotuberculosis. In order to find antibodies specifically binding to Y. pestis we screened a large single chain Fv antibody fragment (scFv) phage display library using purified F1 antigen as a selection target. Different forms of the selected antibodies were used to establish assays for recombinant F1 antigen and Y. pestis detection.MethodsPhage antibody panning was performed against F1 in an automated fashion using the Kingfisher magnetic bead system. Selected scFvs were screened for F1-binding specificity by one-step alkaline phosphatase enzyme linked immunosorbant assay (ELISA), and assayed for binding to recombinant antigen and/or Y. pestis by flow cytometry and whole-cell ELISA.ResultsSeven of the eight selected scFvs were shown to specifically bind both recombinant F1 and a panel of F1-positive Yersinia cells. The majority of the soluble scFvs were found to be difficult to purify, unstable and prone to cross-reactivity with F1-negative Yersinia strains, whereas phage displayed scFvs were found to be easy to purify/label and remarkably stable. Furthermore direct fluorescent labeling of phage displaying scFv allowed for an easy one-step flow cytometry assay. Slight cross-reactivity was observed when fixed cells were used in ELISA.ConclusionsOur high throughput methods of selection and screening allowed for time and cost effective discovery of seven scFvs specifically binding Y. pestis F1 antigen. We describe implementation of different methods for phage-based immunoassay. Based on the success of these methods and the proven stability of phage, we indicate that the use of phage-displayed, rather than phage-free proteins, might generally overcome the shortcomings of scFv antibodies.

Dataset Information

Construction of non-covalent single-chain Fv dimers for hepatocellular carcinoma and their biological functions.

Publications

Construction of non-covalent single-chain Fv dimers for hepatocellular carcinoma and their biological functions.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets