Grafting of material-binding function into antibodies: Functionalization by peptide grafting

https://doi.org/10.1016/j.bbrc.2007.11.062Get rights and content

Abstract

Quite recently, a few antibodies against bulk material surface have been selected from a human repertoire antibody library, and they are attracting immense interest in the bottom-up integration of nanomaterials. Here, we constructed antibody fragments with binding affinity and specificity for nonbiological inorganic material surfaces by grafting material-binding peptides into loops of the complementarity determining region (CDR) of antibodies. Loops were replaced by peptides with affinity for zinc oxide and silver material surfaces. Selection of CDR loop for replacement was critical to the functionalization of the grafted fragments; the grafting of material-binding peptide into the CDR2 loop functionalized the antibody fragments with the same affinity and selectivity as the peptides used. Structural insight on the scaffold fragment used implies that material-binding peptide should be grafted onto the most exposed CDR loop on scaffold fragment. We show that the CDR-grafting technique leads to a build-up creation of the antibody with affinity for nonbiological materials.

Section snippets

Materials and methods

Preparation of Fv fragments and independent VH domain with ZnO- and Ag-binding peptide in CDR. The DNA sequences coding the Fv fragments where CDR loops are replaced by ZnO- or Ag-binding peptide (ZnBP, AgBP, respectively; Table 1), were generated by means of overlap extension polymerase chain reaction [21], from plasmid pKTN2 containing the Fv fragment of mouse anti-hen egg white lysozyme monoclonal antibody HyHEL-10 [22]. The amplified sequences for the VH and VL domains were inserted into

Grafting of ZnO-binding peptide into Fv fragments

Fig. 2 shows the SDS–PAGE analyses of the ZBP-grafted Fv fragments expressed in E. coli. We prepared Fv fragments with ZBP in the CDR2 or CDR3 loop, but not in the CDR1 loop, because the CDR1 loops in both the VH and VL domains contain some of the β-strands that form the immunoglobulin fold. Although grafting ZBP into the CDR loops led to expression of the fragments as insoluble aggregates, all the ZBP-grafted Fv fragments were also present in soluble form in the culture medium and the

Discussion

The VH and VL domains in Fv fragments consist of two β-sheets connected by a disulfide bridge in a sandwich structure, and this framework structure is widely conserved in the antibodies of various mammalian species. The three loops that link the individual β-strands in the frameworks, form the binding pocket in each domain. The first study of grafting into the CDR involved the replacement of the CDR loops in a human antibody with those from a mouse antibody to avoid immunogenicity of the

Acknowledgments

This work was supported by a Scientific Research Grant from the Ministry of Education, Science, Sports, and Culture of Japan (M.U. and I.K.) and by the Industrial Technology Research Grant Program (2005) from the New Energy and Industrial Technology Development Organization (NEDO) of Japan (M.U.). This research was also partly supported by the Hosokawa foundation (M.U.).

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