Optimization of the crystallizability of a single-chain antibody fragment

Single-chain variable antibody fragments (scFvs) are molecules with immense therapeutic and diagnostic potential. Knowledge of their three-dimensional structure is important for understanding their antigen-binding mode as well as for protein-engineering approaches such as antibody humanization. A major obstacle to the crystallization of single-chain variable antibody fragments is their relatively poor homogeneity caused by spontaneous oligomerization. A new approach to optimization of the crystallizability of single-chain variable antibody fragments is demonstrated using a representative single-chain variable fragment derived from the anti-CD3 antibody MEM-57. A Thermofluor-based assay was utilized to screen for optimal conditions for antibody-fragment stability and homogeneity. Such an optimization of the protein storage buffer led to a significantly improved ability of the scFv MEM-57 to yield crystals.

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Fluorophor-linked immunosorbent assay: a time- and cost-saving method for the characterization of antibody fragments using a fusion protein of a single-chain antibody fragment and enhanced green fluorescent protein

Analytical Biochemistry ◽

10.1016/s0003-2697(02)00290-7 ◽

2002 ◽

Vol 309 (1) ◽

pp. 27-34 ◽

Cited By ~ 28

Author(s):

Peter Oelschlaeger ◽

Srividhya Srikant-Iyer ◽

Stefan Lange ◽

Jutta Schmitt ◽

Rolf D Schmid

Keyword(s):

Green Fluorescent Protein ◽

Enhanced Green Fluorescent Protein ◽

Fluorescent Protein ◽

Antibody Fragment ◽

Antibody Fragments ◽

Single Chain ◽

Single Chain Antibody ◽

Green Fluorescent ◽

Single Chain Antibody Fragment

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T-cell activation and cytokine production via a bispecific single-chain antibody fragment targeted to blood-stage malaria parasites

Blood ◽

10.1182/blood-2002-03-0831 ◽

2003 ◽

Vol 101 (6) ◽

pp. 2300-2306 ◽

Cited By ~ 15

Author(s):

Shigeto Yoshida ◽

Tominari Kobayashi ◽

Hiroyuki Matsuoka ◽

Chisato Seki ◽

William L. Gosnell ◽

...

Keyword(s):

T Cells ◽

T Cell Activation ◽

Cell Activation ◽

Recombinant Baculovirus ◽

Antibody Fragment ◽

Antibody Fragments ◽

Therapeutic Drug ◽

Single Chain ◽

Single Chain Antibody ◽

Single Chain Antibody Fragment

A novel bispecific single-chain antibody fragment (biscFv) has been constructed to address the possibility of a new approach to malaria therapeutic drug development. The biscFv consists of 2 different single-chain antibody fragments linked by a flexible peptide linker (Gly4-Ser)3. Of the 2 scFv fragments, one is directed against a conserved epitope of the 19-kDa C-terminal fragment of the major surface protein of human malignant malaria parasite, Plasmodium falciparum, and the other is directed against the CD3 antigen of human T cells. The biscFv expressed by a recombinant baculovirus retained the antigen-binding properties of the corresponding univalent single-chain antibody fragments and formed a bridge between P falciparum and T cells. In cooperation with T cells, the biscFv specifically induced not only interferon γ and tumor necrosis factor α, but also a significant increase of merozoite phagocytosis and growth inhibition of P falciparum in vitro. Thus, the biscFv possesses highly selective malaria-targeting properties and stimulates T cells to induce cytokines, presumably resulting in activation of macrophages, neutrophils, and natural killer cells, and parasite killing in vivo.

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