AsianScientist (Sep. 26, 2017) – In a study published in Structure, a team of researchers in Japan report a hybrid antibody fragment that facilitates the determination of protein structure in X-ray crystallography studies.
Antibodies are proteins that consists of four polypeptide chains: two heavy chains and two light chains joined to form a Y-shaped molecule. An antibody recognizes a specific antigen and is unique to its target because of specialized binding sites located at the upper tips of the Y-shaped complex.
While antibody-based therapeutics have been established as front-line drugs, little headway has been made in the use of antibodies as research tools in small molecule drug discovery, particularly in the field of X-ray crystallography.
X-ray crystallography is a technique that uses X-ray diffraction patterns to determine high-resolution, three-dimensional structures of molecules such as proteins, small organic molecules and materials. The major challenge in X-ray crystallography approaches remains the production of high-quality diffracting crystals.
In recent years, there is increasing use of antibody fragments as crystallization chaperones to aid the structural determination of otherwise ‘uncrystallizable’ or ‘undruggable’ target proteins. This strategy increases the probability of obtaining well-ordered crystals by minimizing the frequent ‘shape-shifting’ of the target protein.
The most commonly used antibody fragment format is the Fab format, but it is difficult to produce in bacterial expression systems because of its large and complex structure. The Fv fragment of the antibody, which contains only one heavy and one light chain, would be ideal for this application thanks to its simple and small architecture. However, Fv is rarely used because the two chains dissociate easily, leading to a loss of function.
In this study, a team of Osaka University researchers has designed a single-chain Fv fragment with improved production compatibility, stability and crystallizability, while maintaining the binding ability of the original molecule.
“We successfully produced a new fragment by fusing an anti-parallel coiled-coil structure derived from a particular domain of a human enzyme, Mst1 kinase, to the antigen-binding sites of an antibody,” said lead author Professor Takao Arimori of Osaka University. “The resulting chimeric molecule, Fv-clasp, was functionally and structurally equivalent to the Fv of the original antibody.”
Notably, switching from Fab to Fv-clasp format markedly enhanced the antibody-assisted crystallization of two biologically important proteins, the extracellular domains of integrin α6β1 and sorLA. Integrin α6β1 plays a significant role in the attachment of induced pluripotent stem cells and embryonic stem cells as well as many cancer cells to the extracellular protein network in tissues. sorLA is protein receptor implicated in Alzheimer’s disease.
“The universal applicability of the Fv-clasp design to large-scale and inexpensive production makes it desirable for industrial applications. Furthermore, its high heat stability is a great advantage for immunotherapies,” said Professor Junichi Takagi of Osaka University who was a co-author of the study.
“Aside from the field of structural biology, we anticipate that the Fv-clasp design will contribute to the expansion of already eminent antibody use in both basic and applied sciences,” he concluded.
The article can be found at: Arimori et al. (2017) Fv-clasp: An Artificially Designed Small Antibody Fragment with Improved Production Compatibility, Stability, and Crystallizability.
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Source: Osaka University.
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with improved production compatibility, stability and crystallizability, while maintaining the binding ability of the original molecule.
