Article

Generation and characterization of recombinant single chain Fv antibody that recognizes platelet glycoprotein Ibalpha.

Thrombosis and Hemostasis Research Unit, Jiangsu Institute of Hematology, The First Affiliated Hospital of Suzhou University, Suzhou 215006, China.
Thrombosis Research (Impact Factor: 3.13). 02/2003; 109(2-3):137-44. DOI: 10.1016/S0049-3848(03)00152-X
Source: PubMed

ABSTRACT A recombinant single chain Fv (scFv) fragment with specific activity against platelet glycoprotein (GP) Ibalpha was developed and characterized. The scFv was generated from the SZ-2 hybridoma, which produced an anti-platelet antibody reactive to GPIbalpha. VH and VL gene segments were generated from the SZ-2 hybridoma by reverse transcribed-polymerase chain reaction (RT-PCR). After cloning into pUCm-T vector, the DNA sequences of both VH and VL genes were analyzed from two different clones, respectively, the same results were obtained. Comparison of SZ-2 variable region to the Kabat database showed that VH belonged to the mouse Ig heavy family XV while VL belonged to the mouse Ig kappa family XXVI. For assembly of the SZ-2 scFv, VH and VL fragments were cloned into pSW1-scFv successively. The scFv was arranged in VH-VL orientation, being joined together with a 15-amino-acid (Gly(4)Ser)(3) linker. The scFv encoding sequence was amplified and cloned into pET22b vector in-frame with a pel B leader sequence to direct secretion of the protein. Escherichia coli strain BL-21(DE3)PlysS was transformed with the recombinant plasmid, and expression of the scFv was induced using isopropyl-beta-D-thiogalactopyranoside (IPTG). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis of the recombinant antibody revealed a protein with apparent molecular weight of approximately 31,000. By comparing band intensity on a Coomassie brilliant blue-stained SDS-PAGE, the production yield of SZ-2 scFv was about 25% of the total cellular proteins. The recombinant SZ-2 scFv antibody was successfully purified using Ni-NTA affinity chromatography with a yield of 120 mg/l. The SZ-2 scFv antibody could bind to platelets demonstrated by enzyme-linked immunosorbent assay (ELISA) and flow cytometry. Analyzed by Western blot, it could bind to platelet GPIb. It retained the binding capacity of its parental SZ-2 monoclonal antibody (MoAb). In functional studies, SZ-2 scFv inhibited platelet agglutination and aggregation induced by ristocetin and thrombin, respectively, but had no effect on ADP-induced platelet aggregation. Therefore, SZ-2 scFv has the potential to be used as an antithrombotic agent.

0 Bookmarks
 · 
40 Views
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Binding of platelet receptor glycoprotein Ibα (GPIbα) to the A1 domain of von Willebrand factor (vWF) is a critical step in both physiologic hemostasis and pathologic thrombosis, for initiating platelet adhesion to subendothelium of blood vessels at sites of vascular injury. Gain-of-function mutations in GPIbα contribute to an abnormally high-affinity binding of platelets to vWF and can lead to thrombosis, an accurate complication causing heart attack and stroke. Of various antithrombotic monoclonal antibodies (mAbs) targeting human GPIbα, 6B4 is a potent one to inhibit the interaction between GPIbα and vWF-A1 under static and flow conditions. Mapping paratope to epitope with mutagenesis experiments, a traditional route in researches of these antithrombotic mAbs, is usually expensive and time-consuming. Here, we suggested a novel computational procedure, which combines with homology modeling, rigid body docking, free and steered molecular dynamics (MD) simulations, to identify key paratope residues on 6B4 and their partners on GPIbα, with hypothesis that the stable hydrogen bonds and salt bridges are the important linkers between paratope and epitope residues. Based on a best constructed model of 6B4 bound with GPIbα, the survival ratios and rupture times of all detected hydrogen bonds and salt bridges in binding site were examined via free and steered MD simulations and regarded as indices of thermal and mechanical stabilizations of the bonds, respectively. Five principal paratope residues with their partners were predicted with their high survival ratios and/or long rupture times of involved hydrogen bonds, or with their hydrogen bond stabilization indices ranked in top 5. Exciting, the present results were in good agreement with previous mutagenesis experiment data, meaning a wide application prospect of our novel computational procedure on researches of molecular of basis of ligand-receptor interactions, various antithrombotic mAbs and other antibodies as well as theoretically design of biomolecular drugs.
    PLoS ONE 01/2012; 7(7):e42263. · 3.73 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: To date, generation of single-chain fragment variable (scFv) has become an established technique used to produce a completely functional antigen-binding fragment in bacterial systems. The advances in antibody engineering have now facilitated a more efficient and generally applicable method to produce Fv fragments. Basically, scFv antibodies produced from phage display can be genetically fused to the marker proteins, such as fluorescent proteins or alkaline phosphatase. These bifunctional proteins having both antigen-binding capacity and marker activity can be obtained from transformed bacteria and used for one-step immunodetection of biological agents. Alternatively, antibody fragments could also be applied in the construction of immunotoxins, therapeutic gene delivery, and anticancer intrabodies for therapeutic purposes. This paper provides an overview of the current studies on the principle, generation, and application of scFv. The potential of scFv in breast cancer research is also discussed in this paper.
    Clinical and Developmental Immunology 01/2012; 2012:980250. · 3.06 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: Binding of platelet glycoprotein Ibα (GPIbα) to von Willebrand factor (vWF) is closely related to the process of physiological hemostasis and pathological thrombosis. Monoclonal antithrombotic antibody SZ2 can inhibit ristocetin- induced platelet agglutination and thrombin-induced platelet aggregation via binding to the anionic region of GPIbα. To investigate the interaction between SZ2 and GPIbα at the residue level, here we perform a computational modeling via homology modeling, molecular docking and intermolecular interactions analysis. Proper conformations of SZ2 and SZ2/GPIbα complex are presented; the key residues in SZ2 and GPIbα are also identified, which are consistent with known experimental evidence. Our study may provide novel insights into SZ2 modification and antibody research, especially in lacking of both crystal structures and mutagenesis data.
    01/2011;